Anti-microbial immunomodulation

ABSTRACT

The invention provides methods of modulating an immune system in a vertebrate host for the therapeutic or prophylactic treatment of infection by a first microbial pathogen in a target tissue, comprising administration at an administration site of an effective amount of an antigenic formulation comprising antigenic determinants specific for a second heterologous microbial pathogen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/308,302 filed Nov. 1, 2016, which application is a U.S. National Phase Application of PCT Application No. PCT/CA2015/050377 filed May 1, 2015, which application claims priority to U.S. Provisional Patent Application Ser. No. 61/988,117 filed May 2, 2014, the disclosure of which are herein incorporated by reference.

FIELD OF THE INVENTION

In various aspects, the invention relates to immunological therapies for treating or preventing pathologies associated with microbial infections in a vertebrate, including the use of microbial vaccines.

BACKGROUND OF THE INVENTION

The innate immune system and the adaptive immune system work in concert in vertebrates to provide, among many other things, protection from pathogenic infection by micro-organisms. Anti-microbial vaccines may be formulated to engage both the innate and adaptive immune systems, but an effective response to vaccination is generally understood to involve a specific adaptive response to one or more of the immunogens present in a vaccine. In this way, multivalent vaccines, such as some pneumococcal vaccines, may be used to elicit a specific adaptive response to more than one serovar. Vaccines have also been described that confer some degree of cross-protective immunity, in which cross-reactivity to an antigen other than the immunogen confers a degree of protective immunity to heterologous microorganisms.

SUMMARY OF THE INVENTION

In one aspect, the invention provides methods and compositions for treating a vertebrate subject for a condition characterized by pathologies associated with a microbial infection, involving the use of microbial vaccines derived from one pathogenic organism to treat infections caused by a heterologous pathogenic organism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C show the number of inflammatory monocytes and dendritic cells in the draining lymph node, lungs, and spleen of mice following treatment with either a K. pneumoniae antigenic composition or PBS, as described in Example 1A herein.

FIG. 2 shows the total number of monocytes and dendritic cells in the lung, peritoneum and spleen of mice following treatment with either a K. pneumoniae antigenic composition, an E. coli antigenic composition, or PBS, as described in Example 1B herein.

FIG. 3 shows the total number of CD4+ T cells, CD8+ T cells, and NK cells from mice treated with either a K. pneumoniae antigenic composition, an E. coli antigenic composition, or PBS, as described in Example 1B herein.

FIGS. 4A and 4B show the total number of (A) inflammatory monocytes and dendritic cells and (B) CD4+ T cells, CD8+ T cells, and NK cells from mice treated with either a heat-inactivated K. pneumoniae antigenic composition, a phenol-inactivated K. pneumoniae antigenic composition, or PBS, as described in Example 10 herein.

FIG. 5 shows the relative frequency of CD11b+ Gr-1+ cells detected from the colons of mice treated with either K. pneumoniae or E. coli antigenic compositions or with PBS control.

FIGS. 6A and 6B show the relative frequency of CD11b+ Gr-1+ cells detected from the lungs of mice treated with either K. pneumoniae or E. coli antigenic compositions or with PBS control.

FIGS. 7A to 7E illustrate microbial prophylaxis as discussed in Example 3, in which treatment with a composition comprising whole killed K. pneumoniae cells provides protective immunity against subsequent challenge by S. pneumoniae. FIG. 7A is a survival curve 5 days post-challenge. FIGS. 7B-7E illustrate S. pneumoniae quantified in nasal wash, lung and spleen homogenate. Megakaryocytes were counted in ten adjacent high power fields (400× magnification) of histological spleen sections. Statistical significance was determined by two-tailed Mann-Whitney test.

FIGS. 8A and 8B illustrate heterologous anti-microbial therapy in a mouse model, as discussed in Example 4, in which treatment with a composition comprising whole killed E. coli cells is effective in ameliorating an infection caused by a heterologous strain of adherent invasive E. coli.

FIGS. 9A and 9B illustrate heterologous anti-microbial therapy in a mouse model, as discussed in Example 3, in which treatment with a composition comprising whole killed Klebsiella pneumonia cells is effective in ameliorating a lung infection caused by Pseudomonas aeruginosa (PA14).

FIG. 10 illustrates heterologous anti-microbial therapy in a mouse model, as discussed in Example 3, in which treatment with a composition comprising whole killed Klebsiella pneumonia cells is effective in ameliorating a lung infection caused by Streptococcus pneumoniae (P1542).

FIGS. 11A and 11B illustrate heterologous anti-microbial therapy in a mouse model, as discussed in Example 5, in which treatment with a composition comprising whole killed E. coli cells is effective in ameliorating a peritoneal infection caused by S. enterica.

FIGS. 12A, 12B and 12C illustrate heterologous anti-microbial therapy in a mouse model, as discussed in Example 5, in which the effectiveness of treatments with two different compositions comprising alternative strains of whole killed E. coli cells is compared to a treatment with an antigenic S. enterica composition in ameliorating a peritoneal infection caused by S. enterica.

FIG. 13 is a bar graph illustrating heterologous anti-microbial therapy in a mouse model, as discussed in Example 6, in which the effectiveness of a S. aureus-derived SSI (QBSAU) is shown against P. aeruginosa challenge in skin, with significantly reduced P. aeruginosa bacterial counts following QBSAU pretreatment.

FIG. 14 is a bar graph illustrating heterologous anti-microbial therapy in a mouse model, as discussed in Example 6, repeating and confirming the data shown in FIG. 13, in which the effectiveness of a S. aureus-derived SSI (QBSAU) is shown against P. aeruginosa challenge in skin, with significantly reduced P. aeruginosa bacterial counts following QBSAU pretreatment.

FIG. 15 is a bar graph illustrating heterologous anti-microbial therapy in a mouse model, as discussed in Example 3c, illustrating Klebsiella pneumoniae SSI (QBKPN) demonstrated statistically superior efficacy in prophylaxis compared to the E. coli SSI (QBECO), in protecting against P. aeruginosa challenge in the lungs.

FIG. 16 is a bar graph illustrating heterologous anti-microbial therapy in a mouse model, as discussed in Example 3c, illustrating Klebsiella pneumoniae SSI (QBKPN) demonstrated statistically superior efficacy in prophylaxis compared to the E. coli SSI (QBECO), in protecting against S. pneumoniae challenge in the lungs.

FIGS. 17A and 17B are two graphs showing targeted heterologous anti-microbial therapy in a geriatric mouse model of lung infection, showing that Klebsiella pneumoniae SSI (QBKPN) protects against S. pneumoniae challenge in lungs of aged mice. Survival benefit for old mice, FIG. 17B, was more pronounced than for young mice, FIG. 17A.

FIG. 18 is a graph illustrating that QBKPN SSI reduces weight loss in aged mice, following challenge with S. pneumoniae, and this benefit is greater for aged mice compared to young mice.

FIG. 19 illustrates antimicrobial prophylaxis in the peritoneal cavity, with a graph illustrating that both QBECO and QBKPN SSIs are protective, as measured by bacterial load in the spleen, with counts in mice treated with QBECO bing significantly lower than mice treated with QBKPN (data are CFU/ml (mean+/− std dev).

DETAILED DESCRIPTION OF THE INVENTION

In various aspects, the invention relates to the surprising discovery that administration of formulations that include antigenic determinants of microbial pathogens that are pathogenic in a particular tissue or organ, is effective in treating pathologies associated with heterologous microbial infections in that specific tissue or organ. Compositions of the invention may for example be administered at a site that is distant from the site of infection. Accordingly, the invention provides antigenic compositions derived from these microbial pathogens, including whole killed bacterial, viral or fungal species, or components thereof, for the therapeutic or prophylactic treatment of heterologous microbial infections, and methods for using the same. The compositions may for example be derived from endogenous pathogens or exogenous pathogens, as described in more detail below.

Antigenic compositions of the invention may be produced that include antigenic determinants that together are specific for or characteristic of a microbial pathogen. In this context, by “specific”, it is meant that the antigenic determinants are sufficiently characteristic of the pathogen that they could be used to raise an immune response, such as an adaptive immune response, against the pathogen in the patient, if the antigenic determinants were to be administered in an appropriate manner to have that effect. It will be recognized that the antigenic determinants need not be so specific that they are characteristic of only one particular strain or species of pathogen, since even a specific immune response against a particular pathogen may be cross reactive with other closely related organisms that are also naturally pathogenic in the tissue or organ in which the heterologous infection is situated and that the antigenic composition is formulated or selected to target.

A “cell” is the basic structural and functional unit of a living organism. In higher organisms, e.g., animals, cells having similar structure and function generally aggregate into “tissues” that perform particular functions. Thus, a tissue includes a collection of similar cells and surrounding intercellular substances, e.g., epithelial tissue, connective tissue, muscle, nerve. An “organ” is a fully differentiated structural and functional unit in a higher organism that may be composed of different types of tissues and is specialized for some particular function, e.g., kidney, heart, brain, liver, etc. Accordingly, by “specific organ, tissue, or cell” is meant herein to include any particular organ, and to include the cells and tissues found in that organ.

“Pathogenic” agents are agents, such as microbes, such as bacteria or viruses, which are known to cause infection in a host in nature, and in this sense, “pathogenic” is used in the context of the present invention to mean “naturally pathogenic”. Although a wide variety of microbes may be capable of causing infection under artificial conditions, such as artificial inoculations of a microbe into a tissue, the range of microbes that naturally cause infection is necessarily limited, and well established by medical practice.

An “infection” is the state or condition in which the body or a part of it is invaded by a pathogenic agent (e.g., a microbe, such as a bacterium) which, under favorable conditions, multiplies and produces effects that are injurious (Taber's Cyclopedic Medical Dictionary, 14th Ed., C. L. Thomas, Ed., F. A. Davis Company, PA, USA). An infection may not always be apparent clinically and may result in only localized cellular injury. Infections may remain subclinical, and temporary if the body's defensive mechanisms are effective. Infections may spread locally to become clinically apparent as an acute, a subacute, or a chronic clinical infection or disease state. A local infection may also become systemic when the pathogenic agent gains access to the lymphatic or vascular system. Infection is usually accompanied by inflammation, but inflammation may occur without infection.

“Inflammation” is the characteristic tissue reaction to injury (marked by swelling, redness, heat, and pain), and includes the successive changes that occur in living tissue when it is injured. Infection and inflammation are different conditions, although one may arise from the other (Taber's Cyclopedic Medical Dictionary, supra). Accordingly, inflammation may occur without infection and infection may occur without inflammation (although inflammation typically results from infection by pathogenic bacteria or viruses). Inflammation is characterized by the following symptoms: redness (rubor), heat (calor), swelling (infection), pain (dolor). Localized visible inflammation on the skin may be apparent from a combination of these symptoms, particularly redness at a site of administration.

Various subjects may be treated in accordance with alternative aspects of the invention. As used herein, a “subject” is an animal, for e.g, a vertebrate such as a mammal, to whom the specific pathogenic bacteria, bacterial antigens, viruses, viral antigens or compositions thereof of the invention may be administered. Accordingly, a subject may be a patient, e.g., a human, suffering from microbial infection, or suspected of having a microbial infection, or at risk for developing a microbial infection. A subject may also be an experimental animal, e.g., an animal model of infection. In some embodiments, the terms “subject” and “patient” may be used interchangeably, and may include a human, a non-human mammal, a non-human primate, a rat, mouse, dog, etc. A healthy subject may be a human who is not suffering from an infection or suspected of having an infection, or who is not suffering from a chronic disorder or condition. A “healthy subject” may also be a subject who is not immunocompromised. By “immunocompromised” or “immunosuppressed” is meant any condition in which the immune system functions in an abnormal or incomplete manner, for example wherein the host is a patient who does not have the ability to respond normally to an infection due to an impaired or weakened immune system. Immunocompromisation or immunosuppression may be due to disease, certain medications (such as chemotherapeutics used in cancer treatment), or conditions present at birth. Immunocompromised subjects may be found more frequently among infants, the elderly, and individuals undergoing extensive drug or radiation therapy. Accordingly, aspects of the invention involve the treatment of pediatric and geriatric patients, or patients at risk of a nosocomial infection. Particular patient populations may for example include patients with compromised immune systems due to HIV infection or AIDS, cancer, solid organ transplantation, stem cell transplantation, sickle cell disease or asplenia, congenital immune deficiencies, chronic inflammatory conditions, cochlear implants, or cerebrospinal fluid leaks.

An “immune response” includes, but is not limited to, one or more of the following responses in a mammal: induction or activation of antibodies, neutrophils, monocytes, macrophages (including both M1-like macrophages and M2-like macrophages as described herein), B cells, T cells (including helper T cells, natural killer cells, cytotoxic T cells, γδ T cells), such as induction or activation by the antigen(s) in an antigenic composition, following administration of the composition. An immune response to a composition thus generally includes the development in the host animal of a cellular and/or antibody-mediated response to the composition of interest. In some embodiments, the immune response is such that it will also result in slowing or stopping the progression of an infection in the animal. An immune response includes both cellular immune responses and humoral immune responses, of both the innate and adaptive immune systems.

In selected embodiments, the methods of the invention may involve determining whether the individual has previously been infected with a pathogen that is pathogenic in the specific organ or tissue; and administering to the individual a therapeutic composition comprising antigenic determinants that are selected or formulated so that together they are specific for the at least one pathogen.

In another aspect, the invention provides methods of formulating compositions of the invention for treating an individual for a condition characterized by microbial disease or infection in a specific organ or tissue. The methods may involve determining whether the individual has previously been infected with at least one pathogen that is pathogenic in the specific organ or tissue; producing an antigenic composition comprising antigenic determinants that together are specific for the at least one pathogen; and formulating the antigenic composition for administration as a therapeutic or anti-microbial composition capable of eliciting an immunological or anti-microbial response in the specific organ or tissue to a heterologous micro-organism.

The methods detailed herein to determining whether a subject has previously been exposed to a pathogen may involve identifying the presence of at least one antibody that recognizes the pathogen. The methods may also or alternatively involve identifying at least one memory B cell that recognizes the pathogen. The methods may also or alternatively involve identifying at least one memory T cell that recognizes the pathogen. The methods may for example involve obtaining the antibody, the memory B cell, or the memory T cell from peripheral circulation or from the targeted specific organ or tissue of the individual.

In another aspect, the invention provides methods of prophylactically treating an individual for an infection in a specific organ or tissue, involving the administration of an infectious micro-organism to provoke a potentiating infection in that organ or tissue. The method may for example involve administering to the individual an infectious dose of at least one pathogen that is pathogenic in the specific organ or tissue, such as an attenuated pathogen; and administering to the individual an anti-microbial composition comprising antigenic determinants, the antigenic determinants selected or formulated so that together they are specific for the at least one pathogen, such as a composition comprising killed whole pathogens, so as to treat or prevent an infection by a heterologous micro-organism. The method may involve these two administration steps occurring simultaneously. The method may involve the second step occurring between 1 hour and 30 days after the first step.

Compositions of the invention may for example be formulated or used for administration at a site that is distinct from the specific organ or tissue that is targeted for treatment, for example by subcutaneous injection or intradermal injection. Compositions may for example be formulated for repeated administration, for example by subcutaneous or intradermal injection. In selected embodiments, compositions of the invention may be formulated or used so as to produce a localized immune response at a site of administration, for example at a site of injection in the skin.

In selected embodiments, pathogens may be selected for use in methods and compositions of the invention on the basis that the pathogen is endogenous to the specific organ or tissue that is targeted for treatment. Alternatively, the pathogen may be exogenous to the specific organ or tissue. The pathogen may be formulated as an attenuated or a killed pathogen, for example to provide an antigenic composition of whole attenuated or killed pathogens. For example, the pathogen may be a bacterium, a virus, a protozoa, a fungus, or a helminth.

In a further aspect, a method of formulating an anti-microbial composition for treating a condition characterized by an infection in a specific organ or tissue is provided. The method involves selecting at least one pathogen that is pathogenic in the specific organ or tissue; producing an antigenic composition comprising antigenic determinants that together are specific for the pathogen; and formulating the antigenic composition for administration as an anti-microbial composition capable of eliciting an anti-microbial response in the specific organ or tissue to a heterologous micro-organism.

The method may further involve a diagnostic step of identifying the specific organ or tissue within which the infection is symptomatic prior to producing the antigenic composition.

Optionally, the antigenic composition may be formulated for subcutaneous injection or intradermal injection. Optionally, the antigenic composition may be formulated for injection to produce a localized skin immune response at a site of administration. Optionally, the method detailed herein is provided such that when a specific tissue or organ is determined, the pathogen is selected from a particular group of pathogens as described herein. In one aspect the pathogen is one that is an endogenous organism, which is a natural cause of infection in the tissue or organ in question. Optionally, the pathogen is an exogenous organism that is a natural cause of infection in the tissue or organ is question, and may include, for example, a bacteria, virus, helminth, or fungus.

Optionally, the antigenic composition may be formulated for repeated subcutaneous or intradermal administration. Optionally, the antigenic composition may be formulated for administration by a route that is not enteric. Optionally, the pathogen detailed herein is a bacteria, a virus, a protozoa, a fungus or a helminth. Further, the method may involve killing or attenuating the pathogen to formulate the antigenic composition as a whole killed or attenuated pathogen composition. The pathogen may be a member of a species of the endogenous flora that is a natural cause of infection in the specific organ or tissue. The pathogen may be an exogenous species that is a natural cause of infection in the specific organ or tissue.

In another aspect, a method of treating an individual for a condition characterized by infection, or a pathology associated with a microbial infection, in a specific organ or tissue is provided. The method involves administering to the individual an anti-microbial composition comprising antigenic determinants. The antigenic determinants are selected or formulated so that together they are specific for at least one pathogen that is pathogenic in the specific organ or tissue. Optionally, the anti-microbial composition may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least two weeks. Further, and without limitation, the dosing may comprise two or more doses (or 10 or more, or 100 or more) over a period from, for example, 1, 2, 3, 4, 5 or 6 days to 1, 2, 3, 4, 5, or 6 weeks.

In another aspect, use of an anti-microbial composition for treating an individual for a condition characterized by inflammation in a specific organ or tissue is disclosed. The anti-microbial composition may for example contain antigenic determinants selected or formulated so that together they are specific for at least one microbial pathogen that is pathogenic in the specific organ or tissue.

In another aspect, use of an anti-microbial composition to formulate a medicament for treating an individual for a condition characterized by pathologies associated with an infection in a specific organ or tissue is disclosed. The anti-microbial composition may for example contain antigenic determinants selected or formulated so that together they are specific for at least one microbial pathogen that is pathogenic in the specific organ or tissue.

In one aspect, a method of comparing immune responses is provided. The method involves administering to an animal having an organ or tissue a medicament having an antigenic composition having antigenic determinants selected or formulated so that together the antigenic determinants are specific for at least one microbial pathogen that is pathogenic in the organ or tissue, extracting a quantifiable immune sample from the organ or tissue, measuring a characteristic of the immune response in the organ or tissue in the quantifiable immune sample following the administration of the medicament, and, comparing the characteristic of the immune response in the quantifiable immune sample to a corresponding characteristic of the immune response in a reference immune sample obtained from a corresponding organ or tissue. Optionally, the reference immune sample may be obtained from the corresponding organ or tissue in the animal prior to the step of administering the medicament. Optionally, the reference immune sample may be obtained from the corresponding organ or tissue in a second animal. Optionally, the animal may have an infection situated in the organ or tissue.

Comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Further, comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. Further and optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. As detailed herein, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, the cytokines are produced as a result of a shift in an activation state of the macrophages. Optionally, the macrophages shift from being M2-like macrophages to being M1-like macrophages. Further and optionally, the macrophages shift from being M1-like macrophages to being M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, the differential gene expression is produced as a result of a shift in an activation state of the macrophages. Optionally, macrophages may shift from being M2-like macrophages to being M1-like macrophages. Further and optionally, the macrophages shift from being M1-like macrophages to being M2-like macrophages.

Optionally, the medicament may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least one week. Optionally, the medicament may be administered intradermally or subcutaneously. Optionally, the medicament may be administered in a dose so that each dose is effective to cause a visible localized inflammatory immune response at the administration site. Optionally, the medicament may be administered so that visible localized inflammation at the administration site occurs within 1 to 48 hours. Further and optionally, the animal may be a mammal. Optionally, the animal may be a human or a mouse.

In another aspect, a method of selecting a therapeutic preparation suitable for treating an individual for an infection in a specific organ or tissue is provided. The method involves providing an animal having an infection situated in a specific organ or tissue, providing a test preparation having one or more antigenic determinants of a microbial pathogen which is pathogenic in the corresponding specific organ or tissue in a healthy individual, measuring a characteristic of the immune response in a reference immune sample obtained from the organ or tissue of the animal, administering the test preparation to the animal, measuring a characteristic of the immune response in a quantifiable immune sample obtained from a corresponding organ or tissue of the animal, comparing the characteristic of the immune response in the in the reference and quantifiable immune samples, and treating an enhanced characteristic of the immune response in the quantifiable immune sample compared to the reference immune sample as an indication of the suitability of the test preparation as a therapeutic preparation. Optionally, the animal is sacrificed before the quantifiable immune sample has been obtained.

Optionally, comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. Further and optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, the cytokines are produced as a result of a shift in an activate state of the macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. Further, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

Further and optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, the differential gene expression may be produced as a result of a shift in an activation state of the macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. Further and optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

In another aspect, a method of selectively targeting an immune response to an infected tissue or an organ in a human subject is provided. The method involves administering to the subject a medicament having an effective amount of a microbial pathogen antigenic composition, wherein the microbial pathogen may be pathogenic in the specific organ or tissue of the subject in which there is an infection caused by a heterologous micro-organism, and the antigenic composition comprises antigenic determinants that together are specific for the microbial pathogen. Optionally, the antigenic composition may include a whole killed bacterial cell composition. Optionally, the medicament may be administered to the subject in an amount and for a time that is effective to up-regulate an anti-microbial immune response in the organ or tissue of the subject in which there is an infection caused by a heterologous micro-organism. Optionally, the method may further involve measuring a characteristic of the immune response. The method also includes prophylactic treatment of infections, by immune-protective vaccination.

In another aspect, a method for treating a human subject for an infection, or a pathology associated with a microbial infection, situated in a tissue or an organ is provided. The method involves administering to the subject a medicament having an effective amount of an antigenic composition comprising a microbial pathogen, such as whole killed bacterial cell or viral compositions, wherein the microbial pathogen is pathogenic in the specific organ or tissue of the subject within which the heterologous microbial infection is situated or within which the future infection is to be prevented. The medicament may be administered to the subject in an amount and for a time that is effective to modulate an immune response in the target organ or tissue. In this way, the invention provides site specific immunomodulators (SSTs), which elicit an immunological response in a target organ or tissue. In select embodiments, the target organ or tissue may be distinct or distant from the site of administration. Optionally, the modulation of the immune response may involve a shift in the activation state of macrophages. Optionally, the modulation of the immune response may involve shifting from a M2-like macrophage response to a M1-like macrophage response. The modulation of the immune response may involve a shift from M1-like macrophages to M2-like macrophages, as those terms are defined herein. Optionally and without limitation, the method may further involve measuring a characteristic of the immune response.

Optionally, comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages. Further and optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. Optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

Further and without limitation, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Further, cytokines may be produced as a result of a shift in an activation state of the macrophages. The macrophages may shift from being M2-like macrophages to being M1-like macrophages. Optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

Further and optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, the differential gene expression may be produced as a result of a shift in an activation state of the macrophages. Further and optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. The macrophages may shift from being M1-like macrophages to being M2-like macrophages.

In another aspect, a method of monitoring efficacy of a treatment regime in an individual being treated for an infection in a specific organ or tissue is provided. The method involves measuring a characteristic of an immune response in a post-treatment immune sample obtained from the specific organ or tissue after the individual has been subject to the treatment regime for a period of time, wherein the presence of a characteristic of the immune response which is greater in magnitude than would be expected had the individual not been subject to the treatment regime, is indicative of the efficacy of the treatment regime; and the treatment regime involves administering a preparation comprising one or more antigenic determinants of a microbial pathogen which is pathogenic in the corresponding specific organ or tissue in a healthy subject.

The method detailed herein may further involve measuring the characteristic of the immune response in a pre-treatment reference sample, wherein the pre-treatment reference sample was obtained from the specific organ or tissue before, at the same time as or after commencement of the treatment regime, but prior to obtaining the post-treatment immune sample, and comparing the characteristic of the immune response in the pre-treatment and post-treatment samples, wherein an increase in the magnitude of the immune response in the post-treatment immune sample compared to the pre-treatment reference sample is indicative of the efficacy of the treatment regime. Optionally, measuring the characteristic of the immune response may involve determining an indication of the number of inflammatory monocytes in a sample of the organ or tissue. Optionally, measuring the characteristic of the immune response may involve determining an indication of the number of macrophages in a sample of the organ or tissue. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages.

As detailed herein in another aspect, the invention also provides methods for formulating an immunogenic composition for treating an infection situated in a specific organ or tissue in a mammal, such as human patient. The method may include selecting at least one microbial pathogen that is naturally pathogenic in the organ or tissue of the mammal within which the heterologous microbial infection is situated. An antigenic composition may be produced that includes antigenic determinants that together are specific for or characteristic of the microbial pathogen.

A diagnostic step may be used to identify the specific organ or tissue within which the infection is situated, prior to producing the antigenic composition targeted to the site of the infection. The site of the infection may be a primary site, or a secondary site of metastasis. The antigenic composition may be sufficiently specific that it would be capable of eliciting an immune response in the mammal specific to the microbial pathogen. The antigenic composition may be a bacterial composition, for example derived from a bacterial species or species that are endogenous to the flora of the patient or from an exogenous species or species. In alternative embodiments, the antigenic composition may be derived from a virus or viruses. Accordingly, the microbial pathogen from which the antigenic composition is derived may be a virus. The microbial pathogen may be killed. In alternative embodiments, the microbial pathogen may be live or attenuated. Immunogenic compositions of the invention may also be formulated or administered with anti-microbial modalities, such as an NSAID. The site of administration may be at a site distant from the site of the infection, for example in an organ or tissue that is not the organ or tissue within which the heterologous infection is situated, for example the skin or subcutaneous tissue.

The antigenic composition may for example be formulated for subcutaneous injection, intradermal injection or oral administration. In embodiments for subcutaneous or intradermal injection, the dosing or formulation of the antigenic composition may be adjusted in order to produce a localized immune reaction visible in the skin at the site of administration, for example an area of inflammation from 2 mm to 100 mm in diameter appearing, for example, 2-48 hours after administration and lasting, for example, 2-72 hours or longer. The antigenic composition may be formulated for repeated subcutaneous or intradermal administration, for example at alternating successive sites.

In some embodiments, the invention involves methods of treating a mammal for an infection situated in a tissue or an organ. In alternative embodiments, the treatment may anticipate the development of the heterologous infection in the tissue, for example if the site of a primary infection suggests the likelihood of the spread of the infection to a particular tissue or organ, then the patient may be prophylactically treated to prevent or ameliorate metastasis to that tissue or organ. The method may include administering to the subject an effective amount of an antigenic composition comprising antigenic determinants that together are specific for at least one microbial pathogen. An aspect of the invention involves the use of a microbial pathogen that is pathogenic in the specific organ or tissue of the mammal within which the heterologous infection is situated. The antigenic composition may be administered, for example by subcutaneous or intradermal injection at an administration site, in successive doses given at a dosage interval, for example of between one hour and one month, over a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1, 2, 3, 4, or 5 years or longer. Each injection dose may for example be metered so that it is effective to cause visible localized inflammation at the administration site, appearing, for example, 1-48 hours after injection.

In another aspect, methods are provided for treating heterologous infections of a specific organ or tissue in a subject by administering one or more antigens of one or more microbial pathogens, such as bacterial, viral or fungal species that are pathogenic in the specific organ or tissue.

In alternative embodiments, the pathogenic microbial species may be capable of causing infection naturally, (i.e., without human intervention) in the specific organ or tissue in a healthy subject, or may have caused an infection in the specific organ or tissue in a healthy subject. In alternative embodiments, the antigen may be administered by administering preparations derived from whole cells of a microbial species. In alternative embodiments, the method may, for example, include administering at least two or more microbial species, or administering at least three or more microbial species, and the microbes may be bacteria or viruses. In alternative embodiments, the method may further include administering a supplement or an adjuvant. An aspect of the invention involves administering antigenic compositions so as to elicit an immune response in said subject.

In alternative embodiments, the microbial pathogen in the antigenic composition may be killed, and thus rendered non-infectious. In some embodiments, the antigenic composition is administered at a site distant from the heterologous infection site, and in selected embodiments of this kind, methods of the invention may be carried out so that they do not produce infection at the heterologous infection site.

As detailed herein, various aspects of the invention involve treating heterologous infections. In this context, treatment may be carried out so as to provide a variety of outcomes. For example, treatment may: provoke an immune reaction that is effective to inhibit or ameliorate the growth or proliferation of an infection; inhibit the growth or proliferation of heterologous micro-organisms; cause remission of an infection; improve quality of life; reduce the risk of recurrence of an infection; inhibit spread of an infection; or, improve patient survival rates in a patient population. In this context, extending the life expectancy of a patient, or patient population, means to increase the number of patients who survive for a given period of time following a particular diagnosis. In some embodiments, treatment may be of patients who have not responded to other treatments, such as patients for whom a chemotherapy or surgery has not been an effective treatment. Treatment in alternative embodiments may for example be before or after onset of heterologous infection. For example prophylactic treatment may be undertaken, for example of patients diagnosed as being at risk of a particular heterologous infection.

Bacteria and Bacterial Colonizations and Infections

Most animals are colonized to some degree by other organisms, such as bacteria, which generally exist in symbiotic or commensal relationships with the host animal. Thus, many species of normally harmless bacteria are found in healthy animals, and are usually localized to the surface of specific organs and tissues. Often, these bacteria aid in the normal functioning of the body. For example, in humans, symbiotic Escherichia coli bacteria may be found in the intestine, where they promote immunity and reduce the risk of infection with more virulent pathogens.

Bacteria that are generally harmless, such as Escherichia coli, can cause infection in healthy subjects, with results ranging from mild to severe infection to death. Whether or not a bacterium is pathogenic (i.e., causes infection) depends to some extent on factors such as the route of entry and access to specific host cells, tissues, or organs; the intrinsic virulence of the bacterium; the amount of the bacteria present at the site of potential infection; or the health of the host animal. Thus, bacteria that are normally harmless can become pathogenic given favorable conditions for infection, and even the most virulent bacterium requires specific circumstances to cause infection. Accordingly, microbial species that are members of the normal flora can be pathogens when they move beyond their normal ecological role in the endogenous flora. For example, endogenous species can cause infection outside of their ecological niche in regions of anatomical proximity, for example by contiguous spread. When this occurs, these normally harmless endogenous bacteria are considered pathogenic.

Specific bacterial species and viruses are known to cause infections in specific cells, tissues, or organs in otherwise healthy subjects. Examples of bacteria and viruses that commonly cause infections in specific organs and tissues of the body are listed below; it will be understood that these examples are not intended to be limiting and that a skilled person would be able to readily recognize and identify infectious or pathogenic bacteria that cause infections, or commonly cause infections, in various organs and tissues in healthy adults (and recognize the relative frequency of infection with each bacterial species) based on the knowledge in the field as represented, for example, by the following publications: Manual of Clinical Microbiology 8th Edition, Patrick Murray, Ed., 2003, ASM Press American Society for Microbiology, Washington D.C., USA; Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases 5th Edition, G. L. Mandell, J. E. Bennett, R. Dolin, Eds., 2000, Churchill Livingstone, Philadelphia, Pa., USA, all of which are incorporated by reference herein.

Infections of the skin are commonly caused by the following bacterial species: Staphylococcus aureus, Beta hemolytic streptococci group A, B, C or G, Corynebacterium diptheriae, Corynebacterium ulcerans, or Pseudomonas aeruginosa; or viral pathogens: rubeola, rubella, varicella-zoster, echoviruses, coxsackieviruses, adenovirus, vaccinia, herpes simplex, or parvo B19.

Infections of the soft tissue (e.g., fat and muscle) are commonly caused by the following bacterial species: Streptococcus pyogenes, Staphylococcus aureus, Clostridium perfringens, or other Clostridium spp.; or viral pathogens: influenza, or coxsackieviruses.

Infections of the breast are commonly caused by the following bacterial species: Staphylococcus aureus, or Streptococcus pyogenes.

Infections of the lymph nodes of the head and neck are commonly caused by the following bacterial species: Staphylococcus aureus, or Streptococcus pyogenes; or viral pathogens: Epstein-Barr, cytomegalovirus, adenovirus, measles, rubella, herpes simplex, coxsackieviruses, or varicella-zoster.

Infections of the lymph nodes of the arm/axillae are commonly caused by the following bacterial species: Staphylococcus aureus, or Streptococcus pyogenes; or viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, adenovirus, or varicella-zoster.

Infections of the lymph nodes of the mediastinum are commonly caused by the following bacterial species: viridans streptococci, Peptococcus spp., Peptostreptococcus spp., Bacteroides spp., Fusobacterium spp., or Mycobacterium tuberculosis; or viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, varicella-zoster, or adenovirus.

Infections of the pulmonary hilar lymph nodes are commonly caused by the following bacterial species: Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenza, Chlamydophila pneumoniae, Bordetella pertussis or Mycobacterium tuberculosis; or viral pathogens: influenza, adenovirus, rhinovirus, coronavirus, parainfluenza, respiratory syncytial virus, human metapneumovirus, or coxsackievirus.

Infections of the intra-abdominal lymph nodes are commonly caused by the following bacterial species: Yersinia enterocolitica, Yersinia pseudotuberculosis, Salmonella spp., Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, or Mycobacterium tuberculosis; or viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, varicella-zoster, adenovirus, influenza, or coxsackieviruses.

Infections of the lymph nodes of the leg/inguinal region are commonly caused by the following bacterial species: Staphylococcus aureus, or Streptococcus pyogenes; or viral pathogens: measles, rubella, Epstein-Barr, cytomegalovirus, or herpes simplex.

Infections of the blood (i.e., septicemia) are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, coagulase-negative staphylococci, Enterococcus spp., Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, Bacteroides fragilis, Streptococcus pneumoniae, or group B streptococci; or viral pathogens: rubeola, rubella, varicella-zoster, echoviruses, coxsackieviruses, adenovirus, Epstein-Barr, herpes simplex, or cytomegalovirus.

Infections of the bone are commonly caused by the following bacterial species: Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, other streptococci spp., Escherichia coli, Pseudomonas spp., Enterobacter spp., Proteus spp., or Serratia spp.; or viral pathogens: parvovirus B19, rubella, or hepatitis B.

Infections of the joint are commonly caused by the following bacterial species: Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, other streptococci spp., Escherichia coli, Pseudomonas spp., Enterobacter spp., Proteus spp., Serratia spp., Neisseria gonorrhea, salmonella species, Mycobacterim tuberculosis, Hemophilus influenza; or viral pathogens: parvovirus B19, rubella, hepatitis B; or fungal pathogen: Scedosporium prolificans

Infections of the meninges are commonly caused by the following bacterial species: Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, or Listeria monocytogenes; or viral pathogens: echoviruses, coxsackieviruses, other enteroviruses, or mumps.

Infections of the brain are commonly caused by the following bacterial species: Streptococcus spp. (including S. anginosus, S. constellatus, S. intermedius), Staphylococcus aureus, Bacteroides spp., Prevotella spp., Proteus spp., Escherichia coli, Klebsiella spp., Pseudomonas spp., Enterobacter spp., or Borrelia burgdorferi; or viral pathogens: coxsackieviruses, echoviruses, poliovirus, other enteroviruses, mumps, herpes simplex, varicella-zoster, flaviviruses, or bunyaviruses.

Infections of the spinal cord are commonly caused by the following bacterial species: Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, Listeria monocytogenes, or Borrelia burgdorferi; or viral pathogens: coxsackieviruses, echoviruses, poliovirus, other enteroviruses, mumps, herpes simplex, varicella-zoster, flaviviruses, or bunyaviruses.

Infections of the eye/orbit are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus milleri, Escherichia coli, Bacillus cereus, Chlamydia trachomatis, Haemophilus influenza, Pseudomonas spp., Klebsiella spp., or Treponema pallidum; or viral pathogens: adenoviruses, herpes simplex, varicella-zoster, or cytomegalovirus.

Infections of the salivary glands are commonly caused by the following bacterial species: Staphylococcus aureus, viridans streptococci (e.g., Streptococcus salivarius, Streptococcus sanguis, Streptococcus mutans), Peptostreptococcus spp., or Bacteroides spp., or other oral anaerobes; or viral pathogens: mumps, influenza, enteroviruses, or rabies.

Infections of the mouth are commonly caused by the following bacterial species: Prevotella melaninogenicus, anaerobic streptococci, viridans streptococci, Actinomyces spp., Peptostreptococcus spp., or Bacteroides spp., or other oral anaerobes; or viral pathogens: herpes simplex, coxsackieviruses, or Epstein-Barr.

Infections of the tonsils are commonly caused by the following bacterial species: Streptococcus pyogenes, or Group C or G B-hemolytic streptococci; or viral pathogens: rhinoviruses, influenza, coronavirus, adenovirus, parainfluenza, respiratory syncytial virus, or herpes simplex.

Infections of the sinuses are commonly caused by the following bacterial species: Streptococcus pneumoniae, Haemophilus influenza, Moraxella catarrhalis, α-streptococci, anaerobic bacteria (e.g., Prevotella spp.), or Staphylococcus aureus; or viral pathogens: rhinoviruses, influenza, adenovirus, or parainfluenza.

Infections of the nasopharynx are commonly caused by the following bacterial species: Streptococcus pyogenes, or Group C or G B-hemolytic streptococci; or viral pathogens: rhinoviruses, influenza, coronavirus, adenovirus, parainfluenza, respiratory syncytial virus, or herpes simplex.

Infections of the thyroid are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, or Streptococcus pneumoniae; or viral pathogens: mumps, or influenza.

Infections of the larynx are commonly caused by the following bacterial species: Mycoplasma pneumoniae, Chlamydophila pneumoniae, or Streptococcus pyogenes; or viral pathogens: rhinovirus, influenza, parainfluenza, adenovirus, corona virus, or human metapneumovirus.

Infections of the trachea are commonly caused by the following bacterial species: Mycoplasma pneumoniae; or viral pathogens: parainfluenza, influenza, respiratory syncytial virus, or adenovirus.

Infections of the bronchi are commonly caused by the following bacterial species: Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bordetella pertussis, Streptococcus pneumoniae, or Haemophilus influenzae; or viral pathogens: influenza, adenovirus, rhinovirus, coronavirus, parainfluenza, respiratory syncytial virus, human metapneumovirus, or coxsackievirus.

Infections of the lung are commonly caused by the following bacterial species: Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, or Haemophilus influenza; or viral pathogens: influenza, adenovirus, respiratory syncytial virus, or parainfluenza.

Infections of the pleura are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Bacteroides fragilis, Prevotella spp., Fusobacterium nucleatum, peptostreptococcus spp., or Mycobacterium tuberculosis; or viral pathogens: influenza, adenovirus, respiratory syncytial virus, or parainfluenza.

Infections of the mediastinum are commonly caused by the following bacterial species: viridans streptococci, Peptococcus spp., Peptostreptococcus spp., Bacteroides spp., Fusobacterium spp., or Mycobacterium tuberculosis; or viral pathogens: measles, rubella, Epstein-Barr, or cytomegalovirus.

Infections of the heart are commonly caused by the following bacterial species: Streptococcus spp. (including S. mitior, S. bovis, S. sanguis, S. mutans, S. anginosus), Enterococcus spp., Staphylococcus spp., Corynebacterium diptheriae, Clostridium perfringens, Neisseria meningitidis, or Salmonella spp.; or viral pathogens: enteroviruses, coxsackieviruses, echoviruses, poliovirus, adenovirus, mumps, rubeola, or influenza.

Infections of the esophagus are commonly caused by the following bacterial species: Actinomyces spp., Mycobacterium avium, Mycobacterium tuberculosis, or Streptococcus spp.; or viral pathogens: cytomegalovirus, herpes simplex, or varicella-zoster.

Infections of the stomach are commonly caused by the following bacterial species: Streptococcus pyogenes or Helicobacter pylori; or viral pathogens: cytomegalovirus, herpes simplex, Epstein-Barr, rotaviruses, noroviruses, or adenoviruses.

Infections of the small bowel are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri; or viral pathogens: adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, or cytomegalovirus.

Infections of the colon/rectum are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri; or viral pathogens: adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, or cytomegalovirus.

Infections of the anus are commonly caused by the following bacterial species: Streptococcus pyogenes, Bacteroides spp., Fusobacterium spp., anaerobic streptococci, Clostridium spp., Escherichia coli, Enterobacter spp., Pseudomonas aeruginosa, or Treponema pallidum; or viral pathogens: herpes simplex.

Infections of the perineum are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterococcus spp., Bacteroides spp., Fusobacterium spp., Clostridium spp., Pseudomonas aeruginosa, anaerobic streptococci, Clostridium spp., or Enterobacter spp.; or viral pathogens: herpes simplex.

Infections of the liver are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Streptococcus (anginosus group), Enterococcus, spp. other viridans streptococci, or Bacteroides spp.; or viral pathogens: hepatitis A, Epstein-Barr, herpes simplex, mumps, rubella, rubeola, varicella-zoster, coxsackieviruses, or adenovirus.

Infections of the gallbladder are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterobacter spp., enterococci, Bacteroides spp., Fusobacterium spp., Clostridium spp., Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri.

Infections of the biliary tract are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterobacter spp., enterococci, Bacteroides spp., Fusobacterium spp., Clostridium spp., Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri; or viral pathogens: hepatitis A, Epstein-Barr, herpes simplex, mumps, rubella, rubeola, varicella-zoster, cocsackieviruses, or adenovirus.

Infections of the pancreas are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterococcus spp., Pseudomonas spp., Staphylococcal spp., Mycoplasma spp., Salmonella typhi, Leptospirosis spp., or Legionella spp.; or viral pathogens: mumps, coxsackievirus, hepatitis B, cytomegalovirus, herpes simplex 2, or varicella-zoster.

Infections of the spleen are commonly caused by the following bacterial species: Streptococcus spp., Staphylococcus spp., Salmonella spp., Pseudomonas spp., Escherichia coli, or Enterococcus spp.; or viral pathogens: Epstein-Barr, cytomegalovirus, adenovirus, measles, rubella, coxsackieviruses, or varicella-zoster.

Infections of the adrenal gland are commonly caused by the following bacterial species: Streptococcus spp., Staphylococcus spp., Salmonella spp., Pseudomonas spp., Escherichia coli, or Enterococcus spp.; or viral pathogens: varicella-zoster.

Infections of the kidney are commonly caused by the following bacterial species: Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providentia spp., Morganella spp., Enterococcus faecalis, or Pseudomonas aeruginosa; or viral pathogens: BK virus, or mumps.

Infections of the ureter are commonly caused by the following bacterial species: Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providentia spp., Morganella spp., or Enterococcus spp.

Infections of the bladder are commonly caused by the following bacterial species: Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providentia spp., Morganella spp., Enterococcus faecalis, or Corynebacterium jekeum; or viral pathogens: adenovirus, or cytomegalovirus.

Infections of the peritoneum are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumonia, Escherichia coli, Klebsiella spp., Proteus spp., enterococci, Bacteroides fragilis, Prevotella melaninogenica, Peptococcus spp., Peptostreptococcus spp., Fusobacterium spp., or Clostridium spp.

Infections of the retroperitoneal area are commonly caused by the following bacterial species: Escherichia coli, or Staphylococcus aureus.

Infections of the prostate are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus mirabilis, enterococci spp., Pseudomonas spp., Corynebacterium spp., or Neisseria gonorrhoeae; or viral pathogens: herpes simplex.

Infections of the testicle are commonly caused by the following bacterial species: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus spp., Streptococcus spp., or Salmonella enteriditis; or viral pathogens: mumps, coxsackievirus, or lymphocytic choriomeningitis virus.

Infections of the penis are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, Neisseria gonorrhoeae, or Treponema pallidum; or viral pathogens: herpes simplex.

Infections of the ovary/adnexae are commonly caused by the following bacterial species: Neisseria gonorrhoeae, Chlamydia trachomatis, Gardenerella vaginalis, Prevotella spp., Bacteroides spp., Peptococcus spp. Streptococcus spp., or Escherichia coli.

Infections of the uterus are commonly caused by the following bacterial species: Neisseria gonorrhoeae, Chlamydia trachomatis, Gardenerella vaginalis, Prevotella spp., Bacteroides spp., Peptococcus spp., Streptococcus spp., or Escherichia coli.

Infections of the cervix are commonly caused by the following bacterial species: Neisseria gonorrhoeae, Chlamydia trachomatis, or Treponema pallidum; or viral pathogens: herpes simplex.

Infections of the vagina are commonly caused by the following bacterial species: Gardenerella vaginalis, Prevotella spp., Bacteroides spp., peptococci spp., Escherichia coli, Neisseria gonorrhoeae, Chlamydia Trachomatis, or Treponema pallidum; or viral pathogens: herpes simplex.

Infections of the vulva are commonly caused by the following bacterial species: Staphylococcus aureus, Streptococcus pyogenes, or Treponema pallidum; or viral pathogens: herpes simplex.

Bacterial Strains/Viral Subtypes

It will be understood by a skilled person in the art that bacterial species are classified operationally as collections of similar strains (which generally refers to groups of presumed common ancestry with identifiable physiological but usually not morphological distinctions, and which may be identified using serological techniques against bacterial surface antigens). Thus, each bacterial species (e.g., Streptococcus pneumoniae) has numerous strains (or serotypes), which may differ in their ability to cause infection or differ in their ability to cause infection in a particular organ/site. For example, although there are at least 90 serotypes of Streptococcus pneumoniae, serotypes 1, 3, 4, 7, 8, and 12 are most frequently responsible for pneumococcal disease in humans.

As a second example, certain strains of Escherichia coli, referred to as extraintestinal pathogenic E. coli (ExPEC), are more likely to cause urinary tract infection or other extraintestinal infections such as neonatal meningitis, whereas other strains, including enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffuse adhering E. coli (DAEC) are more likely to cause gastrointestinal infection/diarrhea. Even among the sub-category of ExPEC strains, specific virulence factors (e.g., production of type-1 fimbriae) enable certain strains to be more capable of causing infection of the bladder, while other virulence factors (e.g., production of P fimbriae) enable other strains to be more capable of causing infection in the kidneys. In accordance with the present invention, an ExPEC strain(s) that is more likely to cause infection in the bladder may be chosen for a formulation to target bladder infection, whereas an ExPEC strain(s) that is more likely to cause infection in the kidney may be chosen for a formulation to target kidney infection. Likewise, one or more of an ETEC, EPEC, EHEC, STEC, EAEC, EIEC or DAEC strains of E. coli (i.e., strains that cause colon infection), may be chosen for a formulation to treat colon infections.

Similarly, there may be numerous subtypes of specific viruses. For example, there are three types of influenza viruses, influenza A, influenza B and influenza C, which differ in epidemiology, host range and clinical characteristics. For example, influenza A is more likely to be associated with viral lung infection, whereas influenza B is more likely to be associated with myositis (i.e., muscle infection). Furthermore, each of these three types of influenza virus have numerous subtypes, which also may differ in epidemiology, host range and clinical characteristics. In accordance with the present invention, one may choose an influenza A subtype most commonly associated with lung infection to target heterologous lung infections, whereas one may choose an influenza B strain most commonly associated with myositis to treat infections of the muscle/soft tissues.

It is understood that a clinical microbiologist skilled in the art would therefore be able to select, based on the present disclosure and the body of art relating to bacterial strains for each species of bacteria (and viral subtypes for each type of virus), the strains of a particular bacterial species (or subtype of a particular virus) to target a specific organ or tissue. In this way, the invention provides site specific immunomodulators (SSTs), in the sense that the formulations and treatments of the invention elicit an immunological response in a target organ or tissue, and that target may be distinct or distant from the site of administration.

Microbial Compositions, Dosages, and Administration

The compositions of the invention include antigens of pathogenic microbial (bacterial or viral) species that are pathogenic in a specific tissue or organ. The compositions may include whole bacterial species, or may include extracts or preparations of the pathogenic bacterial species of the invention, such as cell wall or cell membrane extracts, or whole cells, or exotoxins, or whole cells and exotoxins. The compositions may also include one or more isolated antigens from one or more of the pathogenic bacterial species of the invention; in some embodiments, such compositions may be useful in situations where it may be necessary to precisely administer a specific dose of a particular antigen, or may be useful if administering a whole bacterial species or components thereof (e.g., toxins) may be harmful. Pathogenic bacterial species may be available commercially (from, for example, ATCC (Manassas, Va., USA), or may be clinical isolates from subjects having a bacterial infection of a tissue or organ (e.g., pneumonia).

The microbial compositions of the invention can be provided alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to mammals, for example, humans. As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable for any appropriate form of administration, including subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalational, intratumoral or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound (i.e., the specific bacteria, bacterial antigens, or compositions thereof of the invention), use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to subjects suffering from an infection. Any appropriate route of administration may be employed, for example, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, inhalational, aerosol, topical, intratumoral, sublingual or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; for intranasal formulations, in the form of powders, nasal drops, or aerosols; and for sublingual formulations, in the form of drops, aerosols or tablets.

Methods well known in the art for making formulations are found in, for example, “Remington's Pharmaceutical Sciences” (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. For therapeutic or prophylactic compositions, the pathogenic bacterial species are administered to an individual in an amount effective to stop or slow progression of the infection, or to increase survival of the subject.

An “effective amount” of a pathogenic microbial species or antigen thereof according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction or elimination of the heterologous infection, prevention of microbial infection processes, slowing the growth of the tumour, or an increase in survival time beyond that which is expected using for example the SEER database. A therapeutically effective amount of a pathogenic microbial (bacterial or viral) species or antigen(s) thereof may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the pathogenic bacterial species or virus or antigen thereof are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as prevention of infection, slowing the progress of the infection, reduction or elimination of the heterologous microbial cells.

For administration by subcutaneous or intradermal injection, an exemplary range for therapeutically or prophylactically effective amounts of one or more pathogenic bacterial species may be about 1 million to 100,000 million organisms per ml, or may be 100 million to 7000 million organisms per ml, or may be 500 million to 6000 million organisms per ml, or may be 1000 million to 5000 million organisms per ml, or may be 2000 million to 4000 million organisms per ml, or any integer within these ranges. The total concentration of bacteria per ml may range from 1 million to 100,000 million organisms per ml, or may be 50 million to 7000 million organisms per ml, or may be 100 million to 6000 million organisms per ml, or may be 500 million to 5000 million organisms per ml, or may be 1000 million to 4000 million organisms per ml, or any integer within these ranges. The range for therapeutically or prophylactically effective amounts of antigens of a pathogenic bacterial species may be any integer from 0.1 nM-0.1 M, 0.1 nM-0.05M, 0.05 nM-15 μM or 0.01 nM-10 μM.

It is to be noted that dosage concentrations and ranges may vary with the severity of the condition to be alleviated, or may vary with the subject's immune response. In general, the goal is to achieve an adequate immune response. For administration by subcutaneous or intradermal infection, the extent of an immune response may be determined, for example, by size of delayed local immune skin reaction at the site of injection (e.g., from 0.25 inch to 4 inch diameter). The dose required to achieve an appropriate immune response may vary depending on the individual (and their immune system) and the response desired. Standardized dosages may also be used. In the context of subcutaneous or intradermal administration, if the goal is to achieve a 2 inch local skin reaction, the total bacterial composition dose may, for example, range from 2 million bacteria (e.g., 0.001 ml of a composition with a concentration of 2,000 million organisms per ml) to more than 20,000 million bacteria (e.g., 1 ml of a composition with a concentration of 20,000 million organisms per ml). The concentrations of individual bacterial species or antigens thereof within a composition may also be considered. For example, if the concentration of one particular pathogenic bacterial species, cell size of that species or antigenic load thereof is much higher relative to the other pathogenic bacterial species in the composition, then the local immune skin reaction of an individual may be likely due to its response to this specific bacterial species. In some embodiments, the immune system of an individual may respond more strongly to one bacterial species within a composition than another, depending for example on past history of exposure to infection by a particular species, so the dosage or composition may be adjusted accordingly for that individual. However, in some embodiments detailed herein, an immune response will not be monitored by way of a skin reaction. For example, in some mouse models utilized herein, the effective treatment of such animals with antigenic compositions may not result in corresponding skin reactions. A person skilled in the art will understand that there are alternate ways in which an immune response can be monitored beside relying on the presence or absence of a skin reaction.

For any particular subject, the timing and dose of treatments may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. For example, in the context of subcutaneous or intradermal administration, the compositions may be administered every second day. An initial dose of approximately 0.05 ml may be administered subcutaneously, followed by increases from 0.01-0.02 ml every second day until an adequate skin reaction is achieved at the injection site (for example, a 1 inch to 2 inch diameter delayed reaction of visible redness at the injection site). Once this adequate immune reaction is achieved, this dosing is continued as a maintenance dose. The maintenance dose may be adjusted from time to time to achieve the desired visible skin reaction (inflammation) at the injection site. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1, 2, 3, 4, or 5 years or longer.

Oral dosages may for example range from 10 million to 1,000,000 million organisms per dose, comprising antigenic determinants of one or more species. Oral dosages may be given, for example, from 4 times per day, daily or weekly. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1, 2, 3, 4, or 5 years or longer.

In some embodiments, the invention may include antigenic compositions administered sublingually or by inhalation, or administered to one or more epithelial tissues (i.e., skin by intradermal or subcutaneous injection; lung epithelium by inhalation; gastrointestinal mucosa by oral ingestion; mouth mucosa by sublingual administration) simultaneously or sequentially. Accordingly, in some embodiments the antigenic compositions of the invention are administered so as to provoke an immune response in an epithelial tissue. In some embodiments, one or more epithelial routes of administration may be combined with one or more additional routes of administration, such as intratumoral, intramuscular or intravenous administration.

In various aspects of the invention, the antigenic compositions that are administered to a patient may be characterized as having an antigenic signature, i.e., a combination of antigens or epitopes that are sufficiently specific that the antigenic composition is capable of eliciting an immune response that is specific to a particular pathogen, such as an adaptive immune response. A surprising and unexpected aspect of the invention is that the non-adaptive or non-specific activation of the immune response that is mediated by these specific antigenic compositions is effective to treat heterologous infections situated in the tissues in which the particular pathogen is pathogenic.

Routes of administration and dosage ranges set forth herein are exemplary only and do not limit the route of administration and dosage ranges that may be selected by medical practitioners. The amount of active compound (e.g., pathogenic bacterial species or viruses or antigens thereof) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

In the case of antigenic formulations (i.e. formulations that provoke an immune response), an immunogenically effective amount of a compound or composition of the invention can be provided, alone or in combination with other compounds, such as an immunological adjuvant. The compound may also be linked with a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin to enhance immunogenicity. An antigenic composition is a composition that includes materials that elicit a desired immune response. An antigenic composition may select, activate or expand, without limitation: memory B, T cells, neutrophils, monocytes or macrophages of the immune system to, for example, reduce or eliminate the growth or proliferation of heterologous micro-organisms. In some embodiments, the specific pathogenic microbe, virus, viral antigens, bacteria, bacterial antigens, or compositions thereof of the invention are capable of eliciting the desired immune response in the absence of any other agent, and may therefore be considered to be an antigenic composition. In some embodiments, an antigenic composition includes a suitable carrier, such as an adjuvant, which is an agent that acts in a non-specific manner to increase the immune response to a specific antigen, or to a group of antigens, enabling the reduction of the quantity of antigen in any given dose, or the reduction of the frequency of dosage required to generate the desired immune response. A bacterial antigenic composition may include live or dead bacteria capable of inducing an immune response against antigenic determinants normally associated with the bacteria. In some embodiments, an antigenic composition may include live bacteria that are of less virulent strains (attenuated), and therefore cause a less severe infection. In some embodiments the antigenic composition may include live, attenuated or dead viruses capable of inducing an immune response against antigenic determinants normally associated with the virus.

An antigenic composition comprising killed bacteria for administration by injection may be made as follows. The bacteria may be grown in suitable media, and washed with physiological salt solution. The bacteria may then be centrifuged, resuspended in saline solution, and killed with heat. The suspensions may be standardized by direct microscopic count, mixed in required amounts, and stored in appropriate containers, which may be tested for safety, shelf life, and sterility in an approved manner. In addition to the pathogenic bacterial species and/or antigens thereof, a killed bacterial composition suitable for administration to humans may include 0.4% phenol preservative and/or 0.9% sodium chloride. The bacterial composition may also include trace amounts of brain heart infusion (beef), peptones, yeast extract, agar, sheep blood, dextrose, sodium phosphate and/or other media components.

In some embodiments, the bacterial or microbial composition may be used in tablet or capsule form or drops for oral ingestion, as an aerosol for inhalation, or as drops, aerosol or tablet form for sublingual administration.

In antigenic compositions comprising bacteria, the concentrations of specific bacterial species in compositions for subcutaneous or intradermal injection may be about 1 million to 100,000 million organisms per ml, or may be 100 million to 7000 million organisms per ml, or may be 500 million to 6000 million organisms per ml, or may be 1000 million to 5000 million organisms per ml, or may be 2000 million to 4000 million organisms per ml, or any integer within these ranges. The total concentration of bacteria per ml may range from 1 million to 100,000 million organisms per ml, or may be 50 million to 7000 million organisms per ml, or may be 100 million to 6000 million organisms per ml, or may be 500 million to 5000 million organisms per ml, or may be 1000 million to 4000 million organisms per ml, or any integer within these ranges.

In some embodiments, an antigenic microbial composition for treating an infection at a particular site (e.g., an infection of the lung tissue) may include pathogenic microbes that commonly, more commonly, or most commonly cause infection in that tissue or organ (e.g., infection in the lung tissue i.e., pneumonia).

In general, the pathogenic bacterial species and antigens thereof of the invention should be used without causing substantial toxicity. Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population).

As detailed herein and in an aspect of the invention, a method of comparing or provoking specific immune responses is provided. The method involves administering to an animal having an organ or tissue a medicament having an antigenic composition, as defined herein. The antigenic composition may have antigenic determinants selected or formulated so that together the antigenic determinants are specific for at least one microbial pathogen that is pathogenic in the organ or tissue, extracting a quantifiable immune sample from the organ or tissue, measuring a characteristic of the immune response in the organ or tissue in the quantifiable immune sample following the administration of the medicament, and, comparing the characteristic of the immune response in the quantifiable immune sample to a corresponding characteristic of the immune response in a reference immune sample obtained from a corresponding organ or tissue. As used herein, an immune sample would contain sufficient biological material to determine a characteristic of an immune response. As used herein, a “characteristic” of an immune response can include, without limitation, the particular number of a particular immune cell type (e.g., macrophage), or a particular cellular marker (e.g., upregulation of an integrin) or gene product (e.g., a cytokine). The foregoing is provided as an example and is non-limiting.

Optionally, the reference immune sample may be obtained from the corresponding organ or tissue in the animal prior to the step of administering the medicament. In another aspect, the reference immune sample may be obtained from the corresponding organ or tissue in a second animal such that it is specifically contemplated that at least two animals (i.e., an animal from which a reference immune sample is obtained and a second animal from which a quantifiable immune sample) could be used in the methods described herein. Optionally, the animal may have an infection situated in the organ or tissue.

Comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells as these cells are known to those skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages.

Macrophages can be defined as either “M1-like macrophages” or “M2-like macrophages”. For example, M1-like macrophages are generally understood by those persons skilled in the art to promote a Th1 CD4+ T cell-mediated response (see, for e.g., Biswas and Mantovani (2010), Nature Immunology 10:889-96). Moreover, M1-like macrophages are generally understood to have efficient antigen presentation capacity, and to be proficient at killing intracellular pathogens (for e.g., viruses). Moreover, M1-like macrophages are generally understood to be proficient, at least as compared with M2-like macrophages, in playing an immunological role in tumour destruction. Those skilled in the art will appreciate that there are numerous biological markers which can be employed to differentiate between M1-like macrophages and M2-like macrophages. For example, and as detailed herein, the expression of Nos2 is generally understood to correlate with an M1-like macrophage as compared with an M2-like macrophage (see, for e.g., Laskin et al. (2010) Annual Rev. Pharmacol. Toxicol. 51: 267-288). Further, and for example, M1-like macrophages are generally understood to produce IL-12 and to be effectively activated by IFN-γ through the IFN-γR (Biswas and Mantovain, supra).

In contrast to M1-like macrophages, M2-like macrophages promote a Th2 CD4+ T cell-mediated response (see, generally: Biswas and Mantovani (2010), Nature Immunology 10:889-96). Moreover, M2-like macrophages are generally understood to be effective and encapsulating and clearing extracellular parasites etc. Further, and in comparison to M1-like macrophages, M2-like macrophages are generally understood by those persons skilled in the art as playing a more significant role in immunoregulation both with respect to T_(reg) and B cells (Biswas and Mantovain, supra). Those persons skilled in the art will appreciate that there are numerous biological markers which can be employed to differentiate between M2-like macrophages and M1-like macrophages. For example, and as described herein, a diminished expression of Nos2 will generally be understood to correlate with M2-like macrophages as compared with higher expression being generally found in M1-like macrophages. Further, and as detailed in experiments herein, the expression of CD206 is generally understood as correlating with M2-like macrophages (see, for e.g., Choi et al. (2010) Gastroenterology 138(7) 2399-409). Further, and as detailed in experiments herein, the expression of F4/80 is generally understood to correlate with M2-like macrophages. Further, and for example, M2-like macrophages are generally understood to be effectively activated by IL-4 or by IL-13 through IL-4Rα (Biswas and Mantovain, supra).

Further, comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages. The shift in the activation state of macrophages may optionally be characterized as a shift from M2-like macrophages to M1-like macrophages or vice versa. Those persons skilled in the art will appreciate that there are numerous biological markers that can be employed to monitor the activation of macrophages. As detailed herein, those skilled in the art will appreciate that defining a macrophage as being activated towards either a M1-like phenotype or a M2-like phenotype can be accomplished by choosing markers that are known to associate with either of the respective phenotypes described herein.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. A person skilled in the art will appreciate that there are numerous cell markers (both extracellular and intracellular) that can be selected which can identify an immune response. For example, as described herein, the marker CD206 is generally understood as correlating with M2-like macrophages (see, for e.g., Choi et al. (2010) Gastroenterology 138(7) 2399-409).

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Those persons skilled in the art will appreciate that cytokines refer to small cell-signalling protein molecules and that there are numerous cytokines known in the art. For example, cytokines have been grouped into type 1 and type 2 classifications based on their role in immunological responses. Common type 1 cytokines include IFN-γ and TGF-β. Common type 2 cytokines include, but are not limited to IL-4 and IL-13. Cytokines can be detected by numerous methodologies known to those persons skilled in the art. For example, and as detailed herein, ELISA experiments were utilized to determine cytokine production from lung tissue.

As detailed herein, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages as has been defined herein. Optionally, the cytokines are produced as a result of a shift in an activation state of the macrophages. Optionally, the macrophages shift from being M2-like macrophages to being M1-like macrophages. Further and optionally, the macrophages shift from being M1-like macrophages to being M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. The term “differential gene expression” is understood to mean an appreciable difference between the expression of a particular gene of interest from at least two experimental conditions. For example, if under a first experimental condition a particular gene has a defined expression level as defined by gene expression methods used by those persons skilled in the art and if under a second experimental condition the same gene has an appreciable difference in its expression level, then there is differential expression of the gene of interest. Those persons skilled in the art will understand that there are numerous methodologies with which to detect differential gene expression. For example, commercially available quantitative PCR techniques can be used as detailed herein with respect to determining the relative Nos2/Arg1 ratios. Optionally, the differential gene expression is produced as a result of a shift in an activation state of the macrophages. Optionally, macrophages may shift from being M2-like macrophages to being M1-like macrophages as those terms have been defined herein.

In another embodiment, the medicament may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least one week. Optionally, the medicament may be administered intradermally or subcutaneously. Optionally, the medicament may be administered in a dose so that each dose is effective to cause a visible localized inflammatory immune response at the administration site. Optionally, the medicament may be administered so that visible localized inflammation at the administration site occurs within 1 to 48 hours. However, a visible localized inflammatory immune response may not always be present in all circumstances despite an immune response being initiated. Those skilled in the art will appreciate that there are other methods by which the mounting of an immune response can be monitored. For example, the profile (and relative change in characterization) of immune cells from a subject undergoing an immune reaction can be compared with those from a subject that is not undergoing an immune reaction.

Further and optionally with respect to the methods disclosed herein, the animal may be a vertebrate, such as a mammal. Optionally, the animal may be a human or a mouse.

In another aspect, a method of selecting a therapeutic preparation suitable for treating an individual for an infection in a specific organ or tissue is provided. The method involves providing an animal having an infection situated in a specific organ or tissue, providing a test preparation having one or more antigenic determinants of a microbial pathogen which is pathogenic in the corresponding specific organ or tissue in a healthy individual, measuring a characteristic of the immune response in a reference immune sample obtained from the organ or tissue of the animal, administering the test preparation to the animal, measuring a characteristic of the immune response in a quantifiable immune sample obtained from a corresponding organ or tissue of the animal, comparing the characteristic of the immune response in the in the reference and quantifiable immune samples, and treating an enhanced characteristic of the immune response in the quantifiable immune sample compared to the reference immune sample as an indication of the suitability of the test preparation as a therapeutic preparation. Optionally, the animal is sacrificed before the quantifiable immune sample has been obtained.

Optionally, comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages as those terms have been defined herein. Optionally, comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. Further and optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages as those terms have been defined herein.

Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages as those terms have been defined herein. Optionally, the cytokines are produced as a result of a shift in an activate state of the macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages.

Further and optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages as those terms have been defined herein. Optionally, the differential gene expression may be produced as a result of a shift in an activation state of the macrophages. Optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. Further and optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

In another aspect, a method of selectively targeting an immune response to an infected tissue or an organ in a human subject is provided. The method involves administering to the subject a medicament having an effective amount of a microbial pathogen antigenic composition, wherein the microbial pathogen may be pathogenic in the specific infected organ or tissue of the subject and the antigenic composition comprises antigenic determinants that together are specific for the microbial pathogen. Optionally, the antigenic composition may include a whole killed bacterial cell composition. Optionally, the medicament may be administered to the subject in an amount and for a time that is effective to up-regulate an immune response in the infected organ or tissue of the subject. Optionally, the method may further involve measuring a characteristic of the immune response.

In another aspect, a method for treating a human subject for an infection situated in a tissue or an organ is provided. The method involves administering to the subject a medicament having an effective amount of a microbial pathogen antigenic composition comprising a whole killed bacterial cell composition, wherein the microbial pathogen is pathogenic in the specific organ or tissue of the subject within which the infection is situated. The medicament may be administered to the subject in an amount and for a time that is effective to modulate an immune response. Optionally, the modulation of the immune response may involve a shift in the activation state of macrophages. Optionally, the modulation of the immune response may involve shifting from a M2-like macrophage response to a M1-like macrophage response. The modulation of the immune responses may involve shifting from a M1-like macrophage response to a M2-like macrophage response. Optionally, the method may further involve measuring a characteristic of the immune response.

Optionally, comparing the characteristic of the immune response may involve comparing, in the quantifiable and reference immune samples, an indication of the numbers of any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages as those terms have been defined herein. Optionally, comparing the characteristic of the immune response may involve comparing a shift in an activation state of macrophages. Further and optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. Optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

Further and optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cellular markers on any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages as those terms have been defined herein. Optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, cytokines produced by any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Further, cytokines may be produced as a result of a shift in an activation state of the macrophages. The macrophages may shift from being M2-like macrophages to being M1-like macrophages. Optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

Further and optionally, comparing the characteristic of the immune response may involve identifying, in the quantifiable and reference immune samples, differential gene expression produced by any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, the differential gene expression may be produced as a result of a shift in an activation state of the macrophages. Further and optionally, the macrophages may shift from being M2-like macrophages to being M1-like macrophages. The macrophages may shift from being M1-like macrophages to being M2-like macrophages.

In another aspect, a method of monitoring efficacy of a treatment regime in an individual being treated for an infection in a specific organ or tissue is provided. The method involves measuring a characteristic of an immune response in a post-treatment immune sample obtained from the specific organ or tissue after the individual has been subject to the treatment regime for a period of time, wherein the presence of a characteristic of the immune response which is greater in magnitude than would be expected had the individual not been subject to the treatment regime, is indicative of the efficacy of the treatment regime; and the treatment regime involves administering a preparation comprising one or more antigenic determinants of a microbial pathogen which is pathogenic in the corresponding specific organ or tissue in a healthy subject.

The method detailed herein may further involve measuring the characteristic of the immune response in a pre-treatment reference sample, wherein the pre-treatment reference sample was obtained from the specific organ or tissue before, at the same time as or after commencement of the treatment regime, but prior to obtaining the post-treatment immune sample, and comparing the characteristic of the immune response in the pre-treatment and post-treatment samples, wherein an increase in the magnitude of the immune response in the post-treatment immune sample compared to the pre-treatment reference sample is indicative of the efficacy of the treatment regime. Optionally, measuring the characteristic of the immune response may involve determining an indication of the number of inflammatory monocytes in a sample of the organ or tissue. Optionally, measuring the characteristic of the immune response may involve determining an indication of the number of macrophages in a sample of the organ or tissue. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages.

Optionally, measuring the characteristic of the immune response may involve determining an indication of the number of CD11b+ Gr-1+ cells in a sample of the organ or tissue or determining an indication of the number of dendritic cells in a sample of the organ or tissue. Further and optionally, measuring the characteristic of the immune response may involve determining an indication of the number of CD11c+ MHC class II+ cells in a sample of the organ or tissue or determining an indication of the number of CD4+ T cells in a sample of the organ or tissue or determining an indication of the number of CD8+ T cells in a sample of the organ or tissue.

Optionally, measuring the magnitude of the immune response may involve determining an indication of the number of NK cells in a sample of the organ or tissue. Further and optionally, comparing the characteristic of the immune response may involve identifying, in the reference and immune samples, cellular markers on any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. Optionally, the macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages.

Further and optionally, comparing the characteristic of the immune response may involve identifying, in the reference and immune samples, cytokines produced by any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. Optionally, the cytokines may be produced as a result of a shift in an activation state of the macrophages. The macrophages may shift from being M2-like macrophages to being M1-like macrophages. Further and optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

Optionally, comparing the characteristic of the immune response may involve identifying, in the reference and immune samples, differential gene expression produced by any one or more of the following cells as they are commonly understood to those persons skilled in the art: inflammatory monocytes, macrophages, CD11b+ Gr-1+ cells, dendritic cells, CD11c+ MHC class II+ cells, CD4+ T cells, CD8+ T cells, or NK cells. The macrophages may include any one or more of the following: M1-like macrophages or M2-like macrophages. The differential gene expression may be produced as a result of a shift in an activation state of the macrophages. The macrophages may shift from being M2-like macrophages to being M1-like macrophages. Optionally, the macrophages may shift from being M1-like macrophages to being M2-like macrophages.

The viral pathogen utilized herein may be, without limitation: influenza, adenovirus, respiratory syncytial virus, parainfluenza, monkeypox, herpes simplex virus (1 and 2), Varicella zoster, cytomegalovirus, Epstein-Barr virus, coronavirus, human metapneumovirus, Hendra virus, Nipah virus, Hantavirus, Lassa virus, human T-cell lymphotrophic virus, cocksackievirus, echovirus, enterovirus, or rhinovirus, or any virus that is pathogenic in the lung.

The bacterial pathogen utilized herein may be, without limitation: Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenza, Staphylococcus aureus, Chlamydia pneumoniae, Legionella pneumophila, or Bordatella pertussis or any bacterium that is pathogenic in the lung.

The fungal pathogen utilized herein may be, without limitation: Aspergillus fumigatus, Blastomyces sp., Coccidiodes immitis, Coccidiodes posadasii, Cryptococcus neoformans, Cryptococcus gattii, Fusarium sp., Histoplasma capsulatum, Paecilomyces sp., Paracoccidioides brasiliensis, Penicillium mameffei, Pneumocystis jiroveci, Pseudallescheria boydii, Scedosporium apiospermum, Rhizopus sp., Mucor sp., Absidia sp., Cunninghamella sp., Scedosporium prolificans, Stachybotrys chartarum, Trichoderma longibrachiatium, Trichosporon sp., or any fungus that is pathogenic in the lung.

In various aspects, embodiments of the invention relate to compositions comprising components of organisms that may cause infections of the gastrointestinal tract, so that the organism may be characterized as a pathogen. However, an organism that is in some cases pathogenic may not always cause disease. Most animals are colonized to some degree by other organisms, such as bacteria, which generally exist in symbiotic or commensal relationships with the host animal. Thus, many species of normally harmless bacteria are found in healthy animals, and are usually localized to the surface of specific organs and tissues. Often, these bacteria aid in the normal functioning of the body. For example, in humans, symbiotic Escherichia coli bacteria may be found in the intestine, where they promote immunity and reduce the risk of infection with more virulent pathogens.

Bacteria that are generally harmless, such as Escherichia coli, can cause infection in healthy subjects, with results ranging from mild to severe infection to death. Whether or not an organism, such as a bacterium, is pathogenic (i.e., causes infection) depends to some extent on factors such as the route of entry and access to specific host cells, tissues, or organs; the intrinsic virulence of the bacterium; the amount of the bacteria present at the site of potential infection; or the health of the host animal. Thus, organisms that are normally harmless can become pathogenic given favorable conditions for infection, and even virulent organisms may require specific circumstances to cause infection. Accordingly, organisms that are members of the normal flora can be pathogens when they move beyond their normal ecological role in the endogenous flora. For example, endogenous species can cause infection outside of their ecological niche in regions of anatomical proximity, for example by contiguous spread. When this occurs, and in the context of the present invention, these normally harmless endogenous organisms are considered pathogenic.

Specific organisms, such as bacterial species, viruses, worms, and protozoa are known to cause infections in specific regions of the GIT in otherwise healthy subjects. Examples of organisms that commonly cause infections in specific regions of the GIT are listed below; it will be understood that these examples are not intended to be limiting and that a skilled person would be able to readily recognize and identify infectious or pathogenic organisms that cause infections, or commonly cause infections, in various regions of the GIT in healthy adults, based for example on knowledge about particular patient populations, as represented for example by the following publications: Manual of Clinical Microbiology 8th Edition, Patrick Murray, Ed., 2003, ASM Press American Society for Microbiology, Washington D.C., USA; Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases 5th Edition, G. L. Mandell, J. E. Bennett, R. Dolin, Eds., 2000, Churchill Livingstone, Philadelphia, Pa., USA, all of which are incorporated by reference herein.

Infections of the mouth are commonly caused by the following bacterial species: Prevotella melaninogenicus, anaerobic streptococci, viridans streptococci, Actinomyces spp., Peptostreptococcus spp., or Bacteroides spp., or other oral anaerobes; or viral pathogens: herpes simplex, coxsackieviruses, or Epstein-Barr.

Infections of the esophagus are commonly caused by the following bacterial species: Actinomyces spp., Mycobacterium avium, Mycobacterium tuberculosis, or Streptococcus spp.; or viral pathogens: cytomegalovirus, herpes simplex, or varicella-zoster.

Infections of the stomach are commonly caused by the following bacterial species: Streptococcus pyogenes or Helicobacter pylori; or viral pathogens: cytomegalovirus, herpes simplex, Epstein-Barr, rotaviruses, noroviruses, or adenoviruses.

Infections of the small bowel are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri; or viral pathogens: adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, or cytomegalovirus.

Infections of the colon/rectum are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri; or viral pathogens: adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, or cytomegalovirus.

Infections of the anus are commonly caused by the following bacterial species: Streptococcus pyogenes, Bacteroides spp., Fusobacterium spp., anaerobic streptococci, Clostridium spp., Escherichia coli, Enterobacter spp., Pseudomonas aeruginosa, or Treponema pallidum; or viral pathogens: herpes simplex.

Organisms such as bacteria are often classified operationally as collections of similar strains (which generally refer to groups of presumed common ancestry with identifiable physiological but usually not morphological distinctions, and which may be identified using serological techniques against bacterial surface antigens). Thus, each bacterial species (e.g., Escherichia coli) has numerous strains (or serotypes), which may differ in their ability to cause infection or differ in their ability to cause infection in a particular organ/site. Certain strains of Escherichia coli are more likely to cause gastrointestinal infection/diarrhea, including enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), Shiga toxin-producing E. coli (STEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffuse adhering E. coli (DAEC). In accordance with the present invention, one or more of an ETEC, EPEC, EHEC, STEC, EAEC, EIEC or DAEC strains of E. coli (i.e., strains that cause colon infection), may be chosen for a formulation to treat and a heterologous microbial infection, such as an infection of the GIT, for example an IBD-related microbial infection. For example, a non-ETEC strain of E. coli may be used to prepare an antigenic formulation for treating an infection by an ETEC strain of E. coli. Similarly, non-EPEC, non-EHEC, non-STEC, non-EAEC, non-EIEC or non-DAEC strains of E. coli may be used to formulate an antigenic formulation for treating an infection by, respectively, an EPEC, EHEC, STEC, EAEC, EIEC or DAEC strain of E. coli.

Similarly, there may be numerous subtypes of specific viruses, worms, or protozoa, which are associated with disease in a particular population, and are therefore amenable for use in the present invention.

The compositions of the invention include antigens of organisms that are pathogenic in a specific region of the body, such as the GIT. The compositions may include the components of whole organisms, whole cells or whole virions, or may include extracts or preparations of the organisms, such as cell wall or cell membrane extracts, or exotoxins. The compositions may also include one or more isolated antigens from these organisms. Pathogenic organisms may be available commercially (for example from the American Type Culture Collection, Manassas, Va., USA), or may be clinical isolates from subjects having an infection.

The compositions of the invention derived from pathogens can be provided alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to mammals, for example, humans. As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable for any appropriate form of administration, including subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalational, or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound (i.e., the specific bacteria, bacterial antigens, or compositions thereof of the invention), use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Methods well known in the art for making formulations are found in, for example, “Remington's Pharmaceutical Sciences” (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel. For therapeutic or prophylactic compositions, the formulations may be administered to an individual in an amount effective to prevent, stop or slow progression of a microbial infection.

An “effective amount” of a pathogenic species or antigen thereof according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction or elimination of symptoms of a microbial infection. A therapeutically effective amount of a pathogenic species or antigen(s) thereof may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the pathogenic species or antigen thereof are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as prevention of a microbial infection. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of a microbial infection, so that a prophylactically effective amount may be less than a therapeutically effective amount.

For administration by subcutaneous or intradermal injection, an exemplary range for therapeutically or prophylactically effective amounts of one or more pathogenic bacterial species may be about 1 million to 100,000 million organisms per ml, or may be 100 million to 7000 million organisms per ml, or may be 500 million to 6000 million organisms per ml, or may be 1000 million to 5000 million organisms per ml, or may be 2000 million to 4000 million organisms per ml, or any integer within these ranges. The total concentration of bacteria per ml may range from 1 million to 100,000 million organisms per ml, or may be 50 million to 7000 million organisms per ml, or may be 100 million to 6000 million organisms per ml, or may be 500 million to 5000 million organisms per ml, or may be 1000 million to 4000 million organisms per ml, or any integer within these ranges. The range for therapeutically or prophylactically effective amounts of antigens of a pathogenic bacterial species may be any integer from 0.1 nM-0.1 M, 0.1 nM-0.05M, 0.05 nM-15 μM or 0.01 nM-10 μM.

It is to be noted that dosage concentrations and ranges may vary with the severity of the condition to be alleviated, or may vary with the subject's immune response. In general, the goal is to achieve an adequate immune response. For administration by subcutaneous or intradermal infection, the extent of an immune response may be determined, for example, by size of delayed local immune skin reaction at the site of injection (e.g., from 0.25 inch to 4 inch diameter). The dose required to achieve an appropriate immune response may vary depending on the individual (and their immune system) and the response desired. Standardized dosages may also be used.

In the context of subcutaneous or intradermal administration, if the goal is to achieve a 2 inch local skin reaction, using a bacterial composition, the total dose may, for example, range from 2 million bacteria (e.g., 0.001 ml of a composition with a concentration of 2,000 million organisms per ml) to more than 20,000 million bacteria (e.g., 1 ml of an antigenic composition with a concentration of 20,000 million organisms per ml). The concentrations of individual bacterial species or antigens thereof within a composition may also be considered. For example, if the concentration of one particular pathogenic bacterial species, cell size of that species or antigenic load thereof is much higher relative to the other pathogenic bacterial species in the antigenic composition, then the local immune skin reaction of an individual may be likely due to its response to this specific bacterial species. In some embodiments, the immune system of an individual may respond more strongly to one bacterial species within a composition than another, depending for example on past history of exposure to infection by a particular species, so the dosage or composition may be adjusted accordingly for that individual.

For any particular subject, the timing and dose of treatments may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. For example, in the context of subcutaneous or intradermal administration, the compositions may be administered every second day. An initial dose of approximately 0.05 ml may be administered subcutaneously, followed by increases from 0.01-0.02 ml every second day until an adequate skin reaction is achieved at the injection site (for example, a 1 inch to 2 inch diameter delayed reaction of visible redness at the injection site). Once this adequate immune reaction is achieved, this dosing is continued as a maintenance dose. The maintenance dose may be adjusted from time to time to achieve the desired visible skin reaction (inflammation) at the injection site. Dosing may be for a dosage duration, for example of at least 2 weeks, 2 months, 6 months, 1, 2, 3, 4, or 5 years or longer.

In some embodiments, the invention may include antigenic compositions administered to one or more epithelial tissues by a non-enteric route. For example: to skin by intradermal or subcutaneous injection; to lung epithelium by inhalation. Accordingly, in some embodiments the antigenic compositions of the invention are administered so as to provoke an immune response in a non-enteric tissue, such as an epithelial tissue. In some embodiments, one or more non-enteric routes of administration may be combined with one or more additional routes of administration, such as intramuscular or intravenous administration.

In various aspects of the invention, the antigenic compositions that are administered to a patient may be characterized as having an antigenic signature, i.e., a combination of antigens or epitopes that is sufficiently specific that the antigenic composition is capable of eliciting an immune response that is specific to a particular pathogen, such as an adaptive immune response.

The amount of active compound (e.g., bacterial species, viruses, protozoa or helminths, or antigens thereof) in compositions of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

In the case of antigenic formulations, an immunogenically effective amount of a compound of the invention can be provided, alone or in combination with other compounds, with an immunological adjuvant. The compound may also be linked with a carrier molecule, such as bovine serum albumin or keyhole limpet hemocyanin to enhance immunogenicity. An antigenic composition is a composition that includes materials that elicit a desired immune response. An antigenic composition may select, activate or expand memory B, T cells, neutrophils, monocytes or macrophages of the immune system to, for example, reduce or eliminate the symptoms of the microbial infection. In some embodiments, the specific pathogenic microbe, virus, viral antigens, bacteria, bacterial antigens, or compositions thereof of the invention are capable of eliciting the desired immune response in the absence of any other agent, and may therefore be considered to be an antigenic composition. In some embodiments, an antigenic composition includes a suitable carrier, such as an adjuvant, which is an agent that acts in a non-specific manner to increase the immune response to a specific antigen, or to a group of antigens, enabling the reduction of the quantity of antigen in any given dose, or the reduction of the frequency of dosage required to generate the desired immune response. A bacterial antigenic composition may include live or dead bacteria capable of inducing an immune response against antigenic determinants normally associated with the bacteria. In some embodiments, an antigenic composition may include live bacteria that are of less virulent strains (attenuated), and therefore cause a less severe infection. In some embodiments the antigenic composition may include live, attenuated or dead viruses capable of inducing an immune response against antigenic determinants normally associated with the virus.

An antigenic composition comprising killed organisms for administration by injection may be made as follows. The organism may be grown in suitable media, and washed with physiological salt solution. The organism may then be centrifuged, resuspended in saline solution, and killed with heat. The suspensions may be standardized by direct microscopic count, mixed in required amounts, and stored in appropriate containers, which may be tested for safety, shelf life, and sterility in an approved manner. In addition to the organism and/or antigens thereof, a killed preparation suitable for administration to humans may include phenol preservative (for example 0.4%) and/or sodium chloride (for example on the order of 0.9%). The composition may also for example include trace amounts of brain heart infusion (beef), peptones, yeast extract, agar, sheep blood, dextrose, sodium phosphate and/or other media components.

In some embodiments, the antigenic composition may be used as an aerosol for inhalation.

In general, the compositions of the invention should be used without causing substantial toxicity. Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population).

In some embodiments, bacteria that are members of the endogenous flora of a particular region of the GIT may be used to formulate antigenic compositions of the invention. The rows of Table 1 list a number of bacterial species, together with the biological regions in which each species may form a part of the endogenous flora. For example, Abiotrophia spp. are typically members of the endogenous flora of the mouth.

TABLE 1 Human Bacterial Normal Flora (Endogenous Bacterial Human Pathogens) Duodenum/ Bacterial species Mouth Stomach Jejunum Ileum Colon CFU/mL 10{circumflex over ( )}5 10{circumflex over ( )}2 10{circumflex over ( )}5 10{circumflex over ( )}8 10{circumflex over ( )}11 Abiotrophia spp + Acholeplasma + laidlawii Acidaminococcus + + + + fermentans Acinetobacter spp. + + + + Actinobacillus spp. + Actinobaculum spp. + + + + Actinomyces spp. + + + + Aeromonas spp. + + + Anaerorhabdus + + furcosus Anaerococcus + + hydrogenalis Anaerococcus + + lactolyticus Anaerococcus + + prevotii Atopobium spp. + + + + Bacillus spp. + + Bacteroides caccae + + Bacteroides + + distasonis Bacteroides + + eggerthii Bacteroides fragilis + + Bacteroides merdae + + Bacteroides ovatus + + Bacteroides + + splanchnicus Bacteroides + + thetaiotaomicron Bacteroides + + vulgatus Bifidobacterium + + + adolescentis Bifidobacterium + + + bifidum Bifidobacterium + + + breve Bifidobacterium + + + catenulatum Bifidobacterium + + + + dentium Bifidobacterium + + + longum Bilophila + + + + wadsworthia Burkholderia + + + cepacia Butyrivibrio + + + fibrisolvens Campylobacter + + + concisus Campylobacter + + + curvus Campylobacter + + + gracilis Campylobacter + + + jejuni Campylobacter + + + rectus Campylobacter + + + + showae Campylobacter + sputorum Capnocytophaga + granulosum Capnocytophaga + gingivalis Campylobacter + haemolytica Capnocytophaga + + + + ochracea Capnocytophaga + sputigena Cardiobacterium + hominis Cedecea spp + Centipeda + periodontii Citrobacter freundii + + + Citrobacter koseri + + + Clostridium spp. + + + Corynebacterium + accolens Corynebacterium + afermentans Desulfomonas pigra + + + Dysgonomonas spp. + + + Eikenella corrodens + + + + Enterobacter + + + aerogenes Enterobacter + + + cloacae Enterobacter + + + gergoviae Enterobacter + + + sakazakii Enterobacter + + + taylorae Enterococcus spp. + + + Escherichia coli + + + Escherichia + + + fergusonii Escherichia + + + hermannii Escherichia + + + vulneris Eubacterium spp. + + + + Ewingella + americana Finegoldia magnus + + + Fusobacterium + alocis Fusobacterium + + + gonidiaformans Fusobacterium + + + mortiferum Fusobacterium + + + naviforme Fusobacterium + + + + necrophorum Fusobacterium + + nucleatum Fusobacterium + sulci Fusobacterium + + + russii Fusobacterium + + + varium Gardnerella + + + vaginalis Gemella + haemolysans Gemella + + + + morbillorum Globicatella spp. + + Granulicatella spp. + Haemophilus spp. + Hafnia alvei + + + Helcococcus kunzii Helicobacter spp. + + + Kingella spp. + Klebsiella spp. + + + + Lactobacillus + + + + + acidophilus Lactobacillus breve + Lactobacillus casei + Lactobacillus + + + + + fermentum Lactobacillus + + + + reuteri Lactobacillus + + + + + salivarius Leclercia + + + adecarboxylata Leminorella spp. + + + Leptotrichia + buccalis Megasphaera + + + elsdenii Micrococcus luteus + Micrococcus lylae + Micromonas micros + Mitsuokella + + + multiacidus Mobiluncus curisii + + + Mobiluncus + + + mulieris Moellerella + + + wisconsensis Moraxella + catarrhalis other Moraxella spp. + Morganella + + + morganii Mycoplasma + buccale Mycoplasma + fermentans Mycoplasma + hominis Mycoplasma + lipophilum Mycoplasma orale + Mycoplasma + pneumoniae Mycoplasma + salivarium Pantoea + + + agglomerans Pasteurella + multocida Pediococcus spp. + + Peptoniphilus + + + asaccharolyticus Peptostreptococcus + + + + anaerobus Peptostreptococcus + + + productus Porphyromonas + + + + asaccharolytica Porphyromonas + + catoniae Porphyromonas + + endodontalis Porphyromonas + + gingivalis Prevotella buccae + + Prevotella buccalis + + Prevotella corporis + + Prevotella dentalis + + Prevotella denticola + + Prevotella enoeca + + Prevotella + + heparinolytica Prevotella + + intermedia Prevotella loescheii + + Prevotella + + melaninogenica Prevotella + + nigrescens Prevotella oralis + + Prevotella oris + + Prevotella oulorum + + Prevotella tannerae + + Prevotella veroralis + + Prevotella + + zoogleoformans Propionibacterium + propionicum Proteus mirabilis + + Proteus penneri + + Proteus vulgaris + + Providencia rettgeri + + Providencia stuartii + + + Pseudomonas + + + aeruginosa Retortamonas + + + intestinalis Rothia dentocariosa + Rothia mucilaginosa + Ruminococcus + + + productus Selenomonas spp. + Serratia liquefaciens + + Serratia marcescens + + Serratia odorifera + + Staphylococcus + aureus Staphylococcus + epidermidis Streptococcus + + + agalactiae Streptococcus + + + + anginosus Streptococcus bovis + + + Streptococcus + + + + constellatus Streptococcus + criceti Streptococcus + crista Streptococcus + equisimilis Streptococcus + gordonii Streptococcus + + + intermedius Streptococcus mitis + + Streptococcus + mutans Streptococcus oralis + Streptococcus + parasanguis Streptococcus + + pyogenes Streptococcus + + salivarius Streptococcus + + sanguis Streptococcus + sobrinus Streptococcus + vestibularis Group C + G + + Streptococci Succinivibrio + + + dextrinosolvens Sutterella spp. + + + Suttonella + indologenes Tissierella praeacuta + + + Treponema denticola + Treponema + maltophilum Treponema + socranskii Treponema + vincentii Ureaplasma + urealyticum Veillonella spp. + + + +

Endogenous microbial flora, such as bacteria, have access to tissues for pathogenesis either through contiguous spread or bacteremic spread. Under favorable conditions, all endogenous organisms can become pathogenic and invade locally and spread by contiguous spread to adjacent tissues and organs. Endogenous bacterial flora of the skin, mouth and colon are the species that are understood to also be amenable to bacteremic spread. Bacteria that are members of a particular endogenous flora domain may therefore cause infection in tissues or organs to which these bacteria may spread. Accordingly, one aspect of the invention involves the use of endogenous microbial pathogens to treat a microbial infection having symptoms localized to a region of the GIT in which the endogenous bacteria may spread to cause infection. The columns of Table 2 list domains for endogenous flora. The rows of Table 2 list regions of the GIT within which a microbial infection may be situated. Accordingly, one aspect of the invention involves the use of endogenous microbial pathogens to formulate antigenic compositions, or the selection of existing formulations having the pathogens, for treating a microbial infection situated in the region of the GIT to which the pathogen may spread to cause an infection. Accordingly, in alternative embodiments, a microbial infection that is symptomatic in the region listed in the first column of Table 2 may be treated with antigenic compositions comprising antigenic determinants that are specific for microbial pathogens that are members of the endogenous flora of one or more of the endogenous flora domains listed in the first row of Table 2 and indicated with an X or a check mark in the appropriate row.

TABLE 2 Tissue/Organ Pathogenicity of Endogenous Flora Tissue/organ Duo-denum/ site Mouth Stomach Jejunum Ileum Colon Oral × Tonsil × Nasopharynx/ × Sinus Esophagus × Stomach × Small bowel × × Colon/ × Rectum Anus ×

In accordance with the combined information in Tables 1 and 2, a microbial infection manifest in a particular region of the GIT set out in column 1 of Table 2 may be treated with antigenic compositions comprising antigenic determinants of the corresponding bacterial species of Table 1, so that the column headings in Table 2 are in effect replaced with the bacterial species of Table 1.

In some embodiments, pathogens for use in the invention may be exogenous bacterial pathogens. For example, the organisms listed in Table 3 may be used as microbial pathogens to formulate antigenic compositions, or antigenic compositions having those pathogens may selected, for use to treat a microbial infection situated in the region of the GIT listed with the relevant organism in Table 3. In some embodiments, antigenic determinants of both endogenous and exogenous bacterial species targeted to a specific tissue or organ may be used in combination. For example, an antigenic composition derived from, or specific for, Clostridium difficile, may be used to treat a microbial infection situated in the colon.

TABLE 3 Exogenous Bacterial Human Pathogens, and their Sites of Infection in the GIT Bacterial Species Region of the GIT Aerobacter spp. small bowel, colon, Bacillus anthracis oral, small bowel, colon, hematological Bacillus cereus colon, other Bacillus spp. colon, stomach, small bowel Brucella spp. small bowel, colon Campylobacter coli small bowel, colon Campylobacter jejuni colon Campylobacter small bowel, colon sputorum Clostridium bifermentans small bowel, colon, stomach Clostridium botulinum colon, small bowel Clostridium difficile colon Clostridium indolis small bowel, colon, stomach, Clostridium mangenolii small bowel, colon, stomach Clostridium perfringens small bowel, colon, stomach Clostridium sordellii small bowel, colon, stomach Clostridium sporogenes small bowel, colon, stomach Clostridium small bowel, colon, stomach subterminale Edwarsiella tarda small bowel, colon Francisella tularensis small bowel Helicobacter pylori stomach Leptospirosis spp. oral Listeria monocytogenes small bowel, colon Mycobacterium bovis colon, small bowel Mycobacterium small bowel, colon tuberculosis Pediococcus spp. colon Plesiomonas shigelloides small bowel, colon Rickettsia rickettsiae small bowel Salmonella spp. stomach, small bowel, colon Shigella boydii colon Shigella dysenteriae colon Shigella flexneri colon Shigella sonnei colon other Spirillum spp. colon Streptococcus small bowel zooepidemicus Treponema pallidum oral, anus Tropheryma whipplei small bowel, colon Vibrio cholerae colon, small bowel Vibrio fluvialis small bowel, colon Vibrio furnissii small bowel, colon Vibrio hollisae small bowel, colon Vibrio parahaemolyticus colon, small bowel Yersinia enterocolitica small bowel, colon Yersinia small bowel, colon pseudotuberculosis

In some embodiments, pathogens for use in the invention may be viral pathogens. Table 4 provides an exemplary list of viral pathogens together with the tissue and organ sites for which each viral species is reportedly a pathogen. Accordingly, one aspect of the invention involves utilizing immunogenic compositions that are specific for the named viruses to treat a pathology associated with a heterologous infection situated in the region of the GIT that is identified adjacent to the name of the virus in Table 4.

TABLE 4 Viral Human Pathogens and Their Sites of Infection Virus Region of the GIT Herpes Simplex virus (1 and 2) rectum, anus Cytomegalovirus small bowel, colon/rectum Epstein-Barr virus oral Adenovirus oral, small bowel, colon Human papillomavirus anus, oral Orthoreoviruses small bowel, colon, oral Coltiviruses oral Rotaviruses small bowel, colon Alphaviruses small bowel, colon, Coronaviruses oral, small bowel, colon Toroviruses small bowel, colon Parainfluenza viruses oral Respiratory syncytial virus oral Human metapneumovirus oral, small bowel, colon Vesicular stomatitis virus oral, small bowel, colon Rabies virus oral Influenza virus oral Hantaviruses oral Machupo virus small bowel, colon Junin virus small bowel, colon Poliovirus small bowel, colon Coxsackieviruses small bowel, colon Echoviruses oral, small bowel, colon Hepatitis A virus small bowel, colon Rhinoviruses oral Noroviruses and other small bowel, colon Caliciviruses Astroviruses small bowel, colon Picobirnaviruses small bowel, colon Hepatitis E virus small bowel, colon

The cumulative information in Tables 1 through 4 provides an extensive identification of pathogens that may be used in the formulation of antigenic compositions of the invention, together with an identification of the region of the GIT in which these organisms are pathogenic, and accordingly identifies the region of the GIT in which an infection is situated that may be treated with an antigenic anti-microbial formulation of the invention. Pathogens may be selected from endogenous pathogens or exogenous pathogens.

In some embodiments, the pathogen selected for use in antigenic compositions of the invention may be one that is a common cause of acute infection in the region of the GIT in which the microbial infection to be treated is situated. Table 5 identifies bacterial and viral pathogens of this kind, together with the region of the GIT in which they commonly cause infection. Accordingly, in selected embodiments, a microbial infection, such as an IBD-related infection, residing in a region of the GIT identified in the first column of Table 5 may be treated with an antigenic composition that comprises antigenic determinants for one or more of the pathogenic organisms listed in the second column of Table 5.

TABLE 5 Common causes of acute infection (bacteria and viruses) for selected regions of the GIT Selected regions of the GIT Common Bacterial or Viral Pathogens Oral Prevotella melaninogenicus, anaerobic streptococci, viridans streptococci, Actinomyces spp., Peptostreptococcus spp., Bacteroides spp., and other oral anaerobes herpes simplex, coxsackie viruses, Epstein-Barr Stomach Streptococcus pyogenes, Helicobacter pylori cytomegalovirus, herpes simplex, Epstein-Barr, rotaviruses, noroviruses, adenoviruses Small bowel Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, cytomegalovirus Colon/ Escherichia coli, Clostridium difficile, Bacteroides fragilis, Rectum Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, cytomegalovirus Anus Streptococcus pyogenes, Bacteroides spp., Fusobacterium spp., anaerobic streptococci, Clostridium spp., E. coli, Enterobacter spp., Pseudomonas aeruginosa, Treponema pallidum herpes simplex

The specific organisms which commonly cause infection in a specific region of the GIT may vary by geographical location. Table 5 is thus not an exhaustive list of common pathogens for all geographic locations and population groups. It is understood that a clinical microbiologist skilled in the art could determine the common pathogenic species in a particular geographic area or population group for a specific region of the GIT in accordance with the invention.

Humans are hosts to a wide range of gastrointestinal parasites, including various protozoa and helminths, which for purposes of the present invention constitute pathogens of the GIT (Schafer, T. W., Skopic, A. Parasites of the small intestine. Curr Gastroenterol Reports 2006; 8:312-20; Jernigan, J., Guerrant, R. L., Pearson, R. D. Parasitic infections of the small intestine. Gut 1994; 35:289-93; Sleisenger & Fordtran's Gastrointestinal and liver disease. 8th ed. 2006; Garcia, L. S. Diagnostic medical parasitology. 5th ed. 2007). Compositions of the invention may accordingly include antigenic components of various protozoa, including for example: Giardia lamblia, Cryptosporidium parvum, Cryptosporidium hominus, Isospora belli, Sarcocystis species, Coccidian like bodies (Cyclospora species), Enterocytozoon bieneusi, Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Endolimax nana, Iodamoeba bütschlii, Dientameoba fragilis, Blastocystis hominus, Cyclospora cayetanensis, Microsporidia, Trypanosoma cruzi, Chilomastix mesnili, Pentatrichomonas hominis, Balantidium coli. Similarly, compositions of the invention may include antigenic components of various helminths, including for example: Cestodes (tapeworms), Taenia saginata, Taenia solium, Diphyllobothrium species, Hymenolepis nana, Hymenolepis diminuta, Dipylidium caninum, Nematodes (round worms), Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus, Ancylostoma duodenale, Ancylostoma caninum, Tichuris trichiura, Capillaria philippinensis, Trichostrongylus species, Trichinella species, Necator americanus, Anisakis and related species, Angiostrongylus costaricensis, Enterobius vermicularis, Trematodes (flukes), Fasciolopsis buski, Heterophyes species, Echinostoma species, Clonorchis sinensis, Opisthorchis species, Fasciola species, Metagonimus yokogawi, Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma intercalatum, Echinostoma species and Paragonimus species.

In accordance with the foregoing, in various aspects, the invention may involve the treatment of a microbial infection, such as a microbial infection of the GIT, or an IBD-related microbial infection, with formulations of a pathogen is selected from the group consisting of: Acidaminococcus fermentans; Acinetobacter spp.; Actinobaculum spp.; Actinomyces spp.; Aeromonas spp.; Anaerorhabdus furcosus; Anaerococcus hydrogenalis; Anaerococcus lactolyticus; Anaerococcus prevotii; Atopobium spp.; Bacillus spp.; Bacteroides caccae; Bacteroides distasonis; Bacteroides eggerthii; Bacteroides fragilis; Bacteroides merdae; Bacteroides ovatus; Bacteroides splanchnicus; Bacteroides thetaiotaomicron; Bacteroides vulgatus; Bifidobacterium adolescentis; Bifidobacterium bifidum; Bifidobacterium breve; Bifidobacterium catenulatum; Bifidobacterium dentium; Bifidobacterium longum; Bilophila wadsworthia; Burkholderia cepacia; Butyrivibrio fibrisolvens; Campylobacter concisus; Campylobacter curvus; Campylobacter gracilis; Campylobacter jejuni; Campylobacter rectus; Campylobacter showae; Capnocytophaga ochracea; Cedecea spp; Citrobacter freundii; Citrobacter koseri; Clostridium spp.; Desulfomonas pigra; Dysgonomonas spp.; Eikenella corrodens; Enterobacter aerogenes; Enterobacter cloacae; Enterobacter gergoviae; Enterobacter sakazakii; Enterobacter taylorae; Enterococcus spp.; Escherichia coli; Escherichia fergusonii; Escherichia hermannii; Escherichia vulneris; Eubacterium spp.; Finegoldia magnus; Fusobacterium gonidiaformans; Fusobacterium mortiferum; Fusobacterium naviforme; Fusobacterium necrophorum; Fusobacterium nucleatum; Fusobacterium russii; Fusobacterium varium; Gardnerella vaginalis; Gemella morbillorum; Globicatella spp.; Hafnia alvei; Helicobacter spp.; Klebsiella spp.; Lactobacillus acidophilus; Lactobacillus fermentum; Lactobacillus reuteri; Lactobacillus salivarius; Leclercia adecarboxylata; Leminorella spp.; Megasphaera elsdenii; Mitsuokella multiacidus; Mobiluncus curisii; Mobiluncus mulieris; Moellerella wisconsensis; Morganella morganii; Pantoea agglomerans; Pediococcus spp.; Peptoniphilus asaccharolyticus; Peptostreptococcus anaerobus; Peptostreptococcus productus; Porphyromonas asaccharolytica; Proteus mirabilis; Proteus penneri; Proteus vulgaris; Providencia rettgeri; Providencia stuartii; Pseudomonas aeruginosa; Retortamonas intestinalis; Ruminococcus productus; Serratia liquefaciens; Serratia marcescens; Serratia odorifera; Streptococcus agalactiae; Streptococcus anginosus; Streptococcus bovis; Streptococcus constellatus; Streptococcus intermedius; Group C+G Streptococci; Succinivibrio dextrinosolvens; Sutterella spp.; Tissierella praeacuta; Veillonella spp.; Aerobacter spp.; Bacillus anthracis; Bacillus cereus; other Bacillus spp.; Borrelia recurrentis; Brucella spp.; Campylobacter coli; Campylobacter fetus; Campylobacter jejuni; Campylobacter sputorum; Clostridium bifermentans; Clostridium botulinum; Clostridium difficile; Clostridium indolis; Clostridium mangenolii; Clostridium perfringens; Clostridium sordellii; Clostridium sporogenes; Clostridium subterminale; Edwarsiella tarda; Francisella tularensis; Listeria monocytogenes; Mycobacterium bovis; Mycobacterium tuberculosis; Pediococcus spp.; Plesiomonas shigelloides; Rickettsia rickettsiae; Salmonella spp.; Shigella boydii; Shigella dysenteriae; Shigella flexneri; Shigella sonnei; other Spirillum spp.; Streptococcus zooepidemicus; Tropheryma whipplei; Vibrio cholerae; Vibrio fluvialis; Vibrio furnissii; Vibrio hollisae; Vibrio parahaemolyticus; Yersinia enterocolitica; Yersinia pseudotuberculosis; Herpes Simplex virus (1 and 2); Cytomegalovirus; Adenovirus; Orthoreoviruses; Rotaviruses; Alphaviruses; Coronaviruses; Toroviruses; Human metapneumovirus; Vesicular stomatitis virus; Machupo virus; Junin virus; Poliovirus; Coxsackieviruses; Echoviruses; Hepatitis A virus; Noroviruses and other Caliciviruses; Astroviruses; Picobirnaviruses; and Hepatitis E virus.

In alternative aspects, the invention may involve the treatment of a microbial infection, such as an infection of the GIT, for example an IBD-related infection, with formulations wherein the pathogen is selected from the group of common small and larger bowel pathogens, for example the group consisting of: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri; adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, and cytomegalovirus.

In selected embodiments, the invention involves diagnostic steps to assess a patient's previous exposure to an organism. For example, the diagnostic steps may include taking a medical history of exposure to selected pathogens, and/or evaluating a patient's immune response to a selected pathogen. For example, a serology test may be conducted to detect antibodies to selected pathogens in a patient's sera. In connection with this aspect of the invention, antigenic determinants of a selected pathogen may be chosen for use in an immunogenic composition on a selected patient based on a diagnostic indication that the patient has had one or more prior exposure(s) to the pathogen, for example by virtue of the presence of antibodies to antigenic determinants of that pathogen in the patient's sera.

In further selected embodiments, the invention involves diagnostic steps to assess a patient's immunological response to treatment with a selected immunogenic composition. For example, the diagnostic steps may include evaluating a patient's immune response to the antigenic determinants of that immunogenic composition, for example using a serological test to detect antibodies to those antigenic determinants. In connection with this aspect of the invention a treatment with a selected immunogenic composition may be continued if the evaluation indicates that there is an active immunological response to the antigenic determinants of that composition, and the treatment may be discontinued, and an alternative treatment with a different immunogenic composition may be initiated, if the evaluation indicates that there is not a sufficiently active immunological response to the antigenic determinants of the immunogenic composition.

One aspect of the invention involves the treatment of pathologies associated with microbial lung infections with antigenic compositions that comprise antigenic determinants of microbial pathogens that are known to be lung pathogens, such as exogenous lung pathogens or pathogens that are members of the endogenous flora of the respiratory system. For example, antigenic determinants of the endogenous bacterial respiratory flora species that most commonly cause infection in the lung (see Table 5) may be used to treat infections situated in the lung: Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenza. Similarly, common viral lung pathogens from Table 5 may be selected for use in some embodiments. Alternatively, a more exhaustive list of endogenous lung pathogens may be selected from Table 1, based on the pathogenicity information provided in Table 2. In further alternative embodiments, viral lung pathogens listed in Table 4 may be used. And in further alternative embodiments, exogenous bacterial lung pathogens from Table 3 may be used in formulating antigenic compositions of the invention, i.e. selected from the group consisting of: Achromobacter spp., Actinomadura spp., Alcaligenes spp., Anaplasma spp., Bacillus anthracis, other Bacillus spp., Balneatrix spp., Bartonella henselae, Bergeyella zoohelcum, Bordetella holmesii, Bordetella parapertussis, Bordetella pertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucella spp., Burkholderia gladioli, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter fetus, Capnoctyophaga canimorsus, Capnoctyophaga cynodegmi, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydophila pneumoniae, Chromobacterium violaceum, Chlamydophila psittaci, Chryseobacterium spp., Corynebacterium pseudotuberculosis, Coxiella bumetii, Francisella tularensis, Gordonia spp., Legionella spp., Leptospirosis spp., Mycobacterium avium, Mycobacterium kansasii, Mycobacterium tuberculosis, other Mycobacterium spp., Nocardia spp., Orientia tsutsugamushi, Pandoraea spp., Pseudomonas aeruginosa, other Pseudomonas spp., Rhodococcus spp., Rickettsia conorii, Rickettsia prowazekii, Rickettsia rickettsiae, Rickettsia typhi.

Infections may also arise in bronchial tissue and therefore, in some embodiments, antigenic compositions that comprise antigenic determinants of microbial pathogens that are known to cause bronchial infection may be used to treat patients with infections situated in the bronchial tissue, including, for example, the following common causes of bronchial infection: Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bordetella pertussis, Streptococcus pneumoniae, Haemophilus influenzae, influenza virus, adenovirus, rhinovirus, coronavirus, parainfluenza, respiratory syncytial virus, human metapneumovirus, or coxsackievirus. Infections that are located in both lung and bronchial tissue may be treated with antigenic compositions that comprise antigenic determinants of microbial pathogens that are known to cause both lung and bronchial infection (for example, Streptococcus pneumoniae, Haemophilus influenza and Mycoplasma pneumoniae are all common lung and bronchial pathogens) or alternatively, with antigenic compositions that comprise antigenic determinants of microbial pathogens that are known to cause lung infection and antigenic determinants of microbial pathogens that are known to cause bronchial infection.

One aspect of the invention involves the treatment of pathologies associated with microbial infections of the colon with antigenic compositions that comprise antigenic determinants of heterologous microbial pathogens that are known to be colon pathogens, such as pathogens that are members of the endogenous flora of the colon or exogenous colonic pathogens. For example, antigenic determinants of the following microbial species may be used to treat heterologous infections situated in the colon: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri; adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, or cytomegalovirus. In selected embodiments, antigenic determinants of E. coli, the most common bacterial cause of colon infection, may be used alone or with antigenic determinants of other common pathogens of the colon to treat pathologies associated with infections of the colon, such as pathologies associated with infections caused by heterologous strains of E. coli.

In alternative aspects, the invention utilizes microbial antigens, such as bacterial or viral antigens, to formulate antigenic compositions, where the microbial species is selected on the basis of the tissue or organ within which the microbe is known to cause infections. Bacterial resident flora are the most common bacterial pathogens, accounting for the vast majority of bacterial infections in most animals, including humans. Resident flora can for example infect through primary attachment, or attachment and invasion following mucosa damage, resulting for example from vascular, trauma, chemical insult, or damage resulting from primary infection.

For microbial pathogens, virulence and infection potential is a combination of the ability of the microbe to adhere, to produce enzymes, to survive immunoproducts (complement, antibody) and to survive the microbiocidal activity of macrophage and neutrophils. Some bacteria, including endogenous bacteria, may be sufficiently virulent as to cause monomicrobial infections, while others are more effective with the synergy of polymicrobial infection. In general, it is often not possible to be precise about the specific role of individual microbes within the milieu of mixed infection. As acute infection may, in some cases, provide more optimal immune stimulation, accordingly, in some embodiments, the invention utilizes microbial species that are involved in acute infection.

In some embodiments, bacteria that are members of the endogenous flora of a particular region may be used to formulate antigenic compositions of the invention. The rows of Table 6 list a number of bacterial species, together with the biological regions in which each species may form a part of the endogenous flora. For example, Abiotrophia spp. are typically members of the endogenous flora of the respiratory tract and the mouth. Further and for example, the organisms listed in Table 6 may be used as microbial pathogens to formulate antigenic compositions, or antigenic compositions having those pathogens may be selected, for use to treat heterologous infections, for example as an anti-microbial treatment for heterologous infections situated in the tissues or organs listed with the relevant organism in Table 6.

TABLE 6 Human Bacterial Normal Flora (Endogenous Bacterial Human Pathogens) Duodenum/ GU Bacterial species Respiratory Mouth Stomach Jejunum Ileum Colon System Genital Skin CFU/mL 10{circumflex over ( )}5 10{circumflex over ( )}2 10{circumflex over ( )}5 10{circumflex over ( )}8 10{circumflex over ( )}11 Abiotrophia spp + + Acholeplasma + + laidlawii Acidaminococcus + + + + + fermentans Acinetobacter spp. + + + + + + + + Actinobacillus spp. + + Actinobaculum spp. + + + + Actinomyces spp. + + + + + + + Aerococcus + christensenii Aerococcus viridans + Aerococcus urinae + Aeromonas spp. + + + Alloiococcus otitis + Anaerorhabdus + + furcosus Anaerococcus + + + + hydrogenalis Anaerococcus + + + lactolyticus Anaerococcus + + + prevotii Arcanobacterium spp. + + Atopobium spp. + + + + + Bacillus spp. + + + Bacteroides caccae + + Bacteroides distasonis + + Bacteroides eggerthii + + Bacteroides fragilis + + + + Bacteroides merdae + + Bacteroides ovatus + + Bacteroides + + splanchnicus Bacteroides + + thetaiotaomicron Bacteroides vulgatus + + Bifidobacterium + + + adolescentis Bifidobacterium + + + + + bifidum Bifidobacterium + + + + + breve Bifidobacterium + + + + + catenulatum Bifidobacterium + + + + + + + dentium Bifidobacterium + + + + + longum Bilophila + + + + + + + wadsworthia Brevibacterium casei + Brevibacterium + epidermidis Burkholderia cepacia + + + + Butyrivibrio + + + fibrisolvens Campylobacter + + + + concisus Campylobacter + + + + curvus Campylobacter + + + + gracilis Campylobacter jejuni + + + Campylobacter + + + rectus Campylobacter + + + + + showae Campylobacter + + sputorum Capnocytophaga + + granulosum Capnocytophaga + + gingivalis Campylobacter + + haemolytica Capnocytophaga + + + + + + + ochracea Capnocytophaga + + sputigena Cardiobacterium + + hominis Cedecea spp + Centipeda periodontii + + Citrobacter freundii + + + Citrobacter koseri + + + Clostridium spp. + + + Corynebacterium + + + accolens Corynebacterium + + + afermentans Corynebacterium + amycolatum Corynebacterium + auris Corynebacterium + + diphtheriae Corynebacterium + durum Corynebacterium + glucuronolyticum Corynebacterium + jeikeium Corynebacterium + macginleyi Corynebacterium + matruchotii Corynebacterium + minutissimum Corynebacterium + propinquum Corynebacterium + pseudodiphtheriticum Corynebacterium + riegelii Corynebacterium + simulans Corynebacterium + + striatum Corynebacterium + ulcerans Corynebacterium + + urealyticum Dermabacter + hominis Dermacoccus + nishinomiyaensis Desulfomonas pigra + + + Dysgonomonas spp. + + + Eikenella corrodens + + + + + Enterobacter + + + aerogenes Enterobacter cloacae + + + Enterobacter + + + gergoviae Enterobacter + + + sakazakii Enterobacter + + + taylorae Enterococcus spp. + + + Escherichia coli + + + + + Escherichia + + + fergusonii Escherichia + + + hermannii Escherichia vulneris + + + Eubacterium spp. + + + + + Ewingella americana + + Finegoldia magnus + + + + + Fusobacterium alocis + + Fusobacterium + + + + + gonidiaformans Fusobacterium + + + mortiferum Fusobacterium + + + + + naviforme Fusobacterium + + + + + necrophorum Fusobacterium + + + nucleatum Fusobacterium sulci + + Fusobacterium russii + + + Fusobacterium + + + varium Gardnerella + + + + + vaginalis Gemella haemolysans + + Gemella morbillorum + + + + + Globicatella spp. + + Granulicatella spp. + + Haemophilus spp. + + + Hafnia alvei + + + Helcococcus kunzii + Helicobacter spp. + + + Kingella spp. + + Klebsiella spp. + + + + + Kocuria spp. + Kytococcus + sedentarius Lactobacillus + + + + + + + + acidophilus Lactobacillus breve + + Lactobacillus casei + + + + Lactobacillus + + cellobiosus Lactobacillus + + + + + + + + fermentum Lactobacillus reuteri + + + + Lactobacillus + + + + + + salivarius Lactococcus spp. + + Leclercia + + + adecarboxylata Leminorella spp. + + + Leptotrichia buccalis + + + + Leuconostoc spp. + + Megasphaera + + + elsdenii Micrococcus luteus + + + Micrococcus lylae + + + Micromonas micros + + Mitsuokella + + + multiacidus Mobiluncus curisii + + + + Mobiluncus mulieris + + + + Moellerella + + + wisconsensis Moraxella catarrhalis + + other Moraxella spp. + + + + Morganella + + + morganii Mycoplasma buccale + + Mycoplasma faucium + Mycoplasma + + + fermentans Mycoplasma + + genitalium Mycoplasmaa hominis + + + Mycoplasma + + lipophilum Mycoplasma orale + + Mycoplasma + penetrans Mycoplasma + + pneumoniae Mycoplasma + primatum Mycoplasma + + salivarium Mycoplasma + spermatophilum Neisseria cinerea + Neisseria flavescens + Neisseria lactamica + Neisseria meningitidis + + Neisseria mucosa + Neisseria + polysaccharea Neisseria sicca + Neisseria subflava + Oligella ureolytica + + Oligella urethralis + + Pantoea agglomerans + + + Pastuerella bettyae + + Pasteurella + + multocida Pediococcus spp. + + Peptococcus niger + + + Peptoniphilus + + + + + + asaccharolyticus Peptoniphilus + lacrimalis Peptostreptococcus + + + + + anaerobus Peptostreptococcus + + + productus Peptostreptococcus + + + vaginalis Porphyromonas + + + + + + asaccharolytica Porphyromonas + + + catoniae Porphyromonas + + + endodontalis Porphyromonas + + + gingivalis Prevotella bivia + + Prevotella buccae + + + Prevotella buccalis + + + + + Prevotella corporis + + + Prevotella dentalis + + + Prevotella denticola + + + Prevotella disiens + + Prevotella enoeca + + + Prevotella + + + heparinolytica Prevotella intermedia + + + Prevotella loescheii + + + + + Prevotella + + + + + melaninogenica Prevotella nigrescens + + + Prevotella oralis + + + + + Prevotella oris + + + Prevotella oulorum + + + Prevotella tannerae + + + Prevotella veroralis + + + + + Prevotella + + + zoogleoformans Propionibacterium + acnes Propionibacterium + avidum Propionibacterium + granulosum Propionibacterium + + propionicum Propionferax + innocuum Proteus mirabilis + + + Proteus penneri + + + Proteus vulgaris + + + Providencia rettgeri + + Providencia stuartii + + + Pseudomonas + + + aeruginosa Retortamonas + + + intestinalis Rothia dentocariosa + + Rothia mucilaginosa + + Ruminococcus + + + productus Selenomonas spp. + + Serratia liquefaciens + + Serratia marcescens + + Serratia odorifera + + Staphylococcus + + + + + aureus Staphylococcus + auricularis Staphylococcus + capitis Staphylococcus + caprae Staphylococcus + cohnii Staphylococcus + + + + + epidermidis Staphylococcus + haemolyticus Staphylococcus + hominis Staphylococcus + lugdunensis Staphylococcus + pasteuri Staphylococcus + saccharolyticus Staphylococcus + + saprophyticus Staphylococcus + schleiferia Staphylococcus + simulans Staphylococcus + xylosus Staphylococcus + warneri Streptococcus + + + + + agalactiae Streptococcus + + + + + + + anginosus Streptococcus bovis + + + Streptococcus + + + + + + + constellatus Streptococcus criceti + + Streptococcus crista + + Streptococcus + + equisimilis Streptococcus + + gordonii Streptococcus + + + + + + intermedius Streptococcus mitis + + + Streptococcus mutans + + Streptococcus oralis + + Streptococcus + + parasanguis Streptococcus + pneumoniae Streptococcus + + + + pyogenes Streptococcus + + + salivarius Streptococcus + + + sanguis Streptococcus + + sobrinus Streptococcus + + vestibularis Group C + G + + Streptococci Succinivibrio + + + dextrinosolvens Sutterella spp. + + + + + Suttonella + + indologenes Tissierella praeacuta + + + Treponema denticola + + Treponema + + maltophilum Treponema minutum + Treponema + phagedenis Treponema refringens + Treponema + + socranskii Treponema vincentii + + Turicella otitidis + Ureaplasma + + + urealyticum Veillonella spp. + + + + + Weeksella virosa + +

Endogenous microbial flora, such as bacteria, have access to tissues for pathogenesis either through contiguous spread or bacteremic spread. Under favorable conditions, all endogenous organisms can become pathogenic and invade locally and spread by contiguous spread to adjacent tissues and organs. Endogenous bacterial flora of the skin, mouth and colon are the species that are understood to also be amenable to bacteremic spread. Bacteria that are members of a particular endogenous flora domain may therefore cause infection in tissues or organs to which these bacteria may spread. Accordingly, one aspect of the invention involves the use of endogenous microbial pathogens to treat an infection of a tissue or organ to which the endogenous bacteria may spread to cause infection. The columns of Table 7 list 9 domains for endogenous flora, the: skin, respiratory system, genitals, GU system, mouth, stomach, duodenum/jejunum, ileum and colon. The rows of Table 7 list organs or tissues within which heterologous microbial infections may be situated. Accordingly, one aspect of the invention involves the use of endogenous microbial pathogens to formulate antigenic compositions, or the selection of existing formulations having the pathogens, for treating heterologous microbial infections situated in tissues or organs to which the pathogen may spread to cause an infection. Accordingly, in alternative embodiments, infections situated in the tissues or organs listed in the first column of Table 7 may be treated with antigenic compositions comprising antigenic determinants that are specific for microbial pathogens that are members of the endogenous flora of one or more of the endogenous flora domains listed in the first row of Table 7 and indicated with an X or a check mark in the appropriate row. For example, infections situated in the prostate may be treated with an antigenic composition having antigenic determinants specific for a microbial pathogen or pathogens endogenous to the GU system and/or genital system. A number of the bacterial species that are endogenous to the endogenous flora domains listed in Table 7 are listed, with the corresponding endogenous flora domains, in Table 6. Accordingly, one aspect of the invention involves the treatment of an infection situated in a tissue listed in Table 7 with an antigenic composition comprising antigenic determinants of the bacterial species that are listed in Table 6, where the regions of endogenous flora linked to the site of the infection in Table 7 match the regions of endogenous flora linked to the bacterial species in Table 6. The examples provided in Tables 6 and 7 may be used to formulate antigenic compositions for use in treating heterologous microbial infections, for example as an anti-microbial treatment for heterologous microbial infections in the organs identified in Tables 6 and 7.

TABLE 7 Tissue/Organ Pathogenicity of Endogenous Flora Duo- Tissue/ GU denum/ organ site Skin Respiratory Genital System Mouth Stomach Jejunum Ileum Colon Skin × × Soft tissue × (i.e. fat and muscle) (e.g., sarcoma) Breast × × Lymph × × × nodes: head and neck Lymph × ✓ ✓ nodes: axillae/arm Lymph × ✓ ✓ nodes: mediastinal Lymph × nodes: pulmonary hilum Lymph × ✓ × × × × nodes: intra- abdominal Lymph × × ✓ ✓ nodes: inguinal/ leg Hematological ✓ ✓ ✓ (e.g. leukemias, multiple myeloma) Bone × ✓ ✓ Joints × ✓ ✓ ✓ Meninges × × Brain ✓ ✓ ✓ Spinal cord ✓ ✓ ✓ Eye/Orbit × × × × Salivary × glands Oral × Tonsil × × Nasopharynx/ × × Sinus Thyroid ✓ ✓ ✓ Larynx × × Lung/Trachea/ × Bronchi Pleura ✓ × ✓ ✓ Mediastinum × Heart ✓ ✓ □ Esophagus × Stomach × Small bowel × × Colon/ × Rectum Anus × × Perineum × × Liver ✓ ✓ ✓ Gallbladder × Biliary tract × Pancreas × Spleen ✓ ✓ ✓ Adrenal ✓ ✓ ✓ gland Kidney ✓ × ✓ ✓ Ureter × Bladder ✓ × × Peritoneum × × × × Retroperitoneal × × × × × area Prostate × × Testicle × × Penis × × × Ovary/ × × × Adnexae Uterus × × × Cervix × × × Vagina × × Vulva × × * Bacteria have access to tissues/organs either through: Contiguous spread (×) or Bacteremic spread: (✓).

In accordance with the combined information in Tables 6 and 7, infections located in the tissues or organs set out in column 1 of Table 7 may be treated with antigenic compositions comprising antigenic determinants of the corresponding but heterologous bacterial species of Table 6, so that the column headings in Table 7 are in effect replaced with the bacterial species of Table 6.

In some embodiments, microbial pathogens for use in the invention may be exongenous bacterial pathogens. For example, the organisms listed in Table 8 may be used as microbial pathogens to formulate antigenic compositions, or antigenic compositions having those pathogens may be selected, for use to treat pathologies associated with heterologous infections situated in the tissues or organs listed with the relevant organism in Table 8. In some embodiments, antigenic determinants of both endogenous and exogenous bacterial species targeted to a specific tissue or organ may be used in combination. For example, an antigenic composition derived from, or specific for, Clostridium difficile, may be used to treat pathologies associated with heterologous microbial infections in the colon.

TABLE 8 Exogenous Bacterial Human Pathogens, and their Sites of Infection bacterial species tissue/organ sites Achromobacter hematological, skin, soft tissue, lung/trachea/bronchi, spp. peritoneum, meninges, bile duct, gallbladder, kidney, bladder, ureter Actinomadura skin, soft tissue, lung/trachea/bronchi, mediastinum, brain, spp. spinal cord, hematological, meninges, joints Aerobacter spp. small bowel, colon, hematological, peritoneum Aerococcus spp. hematological, heart, bone, kidney, bladder, ureter, meninges Alcaligenes spp. lung/trachea/bronchi Anaplasma spp. meninges, hematological, liver, spleen, bone, lung/trachea/bronchi Bacillus anthracis lung/trachea/bronchi, lymph nodes pulmonary hilum, mediastinum, meninges, skin, nasopharynx, tonsil, oral, small bowel, colon, hematological Bacillus cereus colon, eye, hematological other Bacillus hematological, bone, meninges, brain, heart, spp. lung/trachea/bronchi, mediastinum, skin, soft tissue, colon, stomach, small bowel, eye Balneatrix spp. lung/trachea/bronchi, meninges, hematological Bartonella skin, hematological, liver, muscle, lymph nodes, joints bacilliformis Bartonella brain, spinal cord, hematological, skin, liver, bone, pleura, henselae lung/trachea/bronchi, mediastinum, axillary and inguinal lymph nodes, eye, joints Bartonella skin, hematological, liver, spleen, joints quintana Bergeyella skin, soft tissue, meninges, hematological, zoohelcum lung/trachea/bronchi Bordetella lung/trachea/bronchi, hematological holmesii Bordetella nasopharynx, tonsil, lung/trachea/bronchi parapertussis Bordetella nasopharynx, tonsil, lung/trachea/bronchi pertussis Borrelia meninges, brain, spinal cord, skin, eye, hematological, burgdorferi inguinal/axillary/cervical lymph nodes, muscle, liver, spleen, nasopharynx, lung/trachea/bronchi, testes, joints Borrelia brain, spinal cord, hematological, small bowel, liver, recurrentis spleen, salivary glands, lung/trachea/bronchi, lymph nodes, eye, skin Brevundimonas peritoneum, hematological, skin, soft tissue spp. Brucella spp. lung/trachea/bronchi, lymph nodes pulmonary hilum, meninges, brain, spinal cord, lymph nodes, mediastinum, bone, eye, small bowel, colon, liver, biliary tract, kidney, ureter, bladder, hematological, skin, testes, spleen, prostate, joints Burkholderia hematological, meninges, lung/trachea/bronchi gladioli Burkholderia lung/trachea/bronchi, skin, soft tissue, liver, spleen, mallei muscle, lymph nodes pulmonary hilum, mediastinal lymph nodes, mediastinum, head and neck lymph nodes, hematological Burkholderia lung/trachea/bronchi, skin, kidney, bladder, ureter, soft pseudomallei tissue, bone, brain, spinal cord, muscle, hematological, prostate, kidney, ureter, meninges Calymmatobacterium skin, penis, vulva, soft tissue, vagina, cervix, bone, granulomatis hematological, inguinal lymph nodes Campylobacter small bowel, colon coli Campylobacter lung/trachea/bronchi, small bowel, colon, meninges, brain, fetus peritoneum, bone, gallbladder, ovaries, hematological, heart, kidney, bladder, ureter Campylobacter colon, hematological, gallbladder, pancreas, bladder, jejuni bone, meninges Campylobacter small bowel, colon sputorum Capnoctyophaga skin, soft tissue, meninges, hematological, bone, canimorsus lung/trachea/bronchi, eye Capnoctyophaga skin, soft tissue, meninges, hematological, bone, cynodegmi lung/trachea/bronchi, eye CDC groups EF- hematological, eye, skin, soft tissue 4a and EF-4b Chlamydia lung/trachea/bronchi, lymph nodes pulmonary hilum, liver, pneumoniae brain, meninges, skin, thyroid, pancreas, hematological Chlamydia psittaci lung/trachea/bronchi, lymph nodes pulmonary hilum, mediastinum, liver, brain, meninges, hematological, skin, thyroid, pancreas Chlamydia inguinal lymph nodes, penis, vulva, vagina, cervix, uterus, trachomatis ovaries and adnexae, peritoneum, prostate, eye Chlamydophila laryngx, trachea/bronchi, hematological pneumoniae Chromobacterium hematological, liver, spleen, lung/trachea/bronchi, kidney, violaceum bladder, ureter, eye/orbit, bone, brain, meninges, spinal cord Chlamydophila lung/trachea/bronchi psittaci Chryseobacterium meninges, lung/trachea/bronchi, hematological spp. Clostridium small bowel, colon, stomach, skin, soft tissue, bifermentans hematological Clostridium colon, small bowel, skin botulinum Clostridium colon difficile Clostridium indolis small bowel, colon, stomach, skin, soft tissue, hematological Clostridium small bowel, colon, stomach, skin, soft tissue, mangenolii hematological Clostridium small bowel, colon, stomach, skin, soft tissue, perfringens hematological, heart Clostridium small bowel, colon, stomach, skin, soft tissue, sordellii hematological Clostridium small bowel, colon, stomach, skin, soft tissue, sporogenes hematological Clostridium small bowel, colon, stomach, skin, soft tissue, subterminale hematological Clostridium tetani skin, soft tissue Comamonas spp. hematological, peritoneum, eye Corynebacterium neck/axillary/inguinal/mediastinal lymph nodes, lymph pseudotuberculosis nodes pulmonary hilum, lung/trachea/bronchi, mediastinum Coxiella burnetii lung/bronchi/trachea, brain, spinal cord, liver, bone, joints Edwarsiella tarda skin, soft tissue, liver, meninges, small bowel, colon, bone, uterus, ovaries Ehrlichia spp. meninges, brain, spinal cord, hematological, bone, liver, kidney, spleen, lymph nodes Erysipelothrix skin, hematological, bone, brain, peritoneum rhusiopathiae Francisella nasopharynx, oral, tonsil, lung/trachea/bronchi, skin, tularensis axillary/head and neck/inguinal lymph nodes, hematological, eye, small bowel Fusobacterium skin, soft tissue, hematological spp. Gordonia spp. skin, soft tissue, lung/trachea/bronchi, mediastinum, brain, spinal cord, hematological, meninges, eye Haemophilus skin, inguinal lymph nodes, penis, vulva, vagina ducreyi Helicobacter stomach pylori Legionella spp. lung/trachea/bronchi, lymph nodes pulmonary hilum, hematological, brain, spinal cord, muscle, pancreas Leptospirosis spp. lung/trachea/bronchi, pancreas, meninges, brain, spinal cord, skin, lymph nodes, eye, hematological, nasopharynx, oral, tonsil, kidney, liver, spleen Listeria Hematological (septicemia), brain, meninges, spinal cord, monocytogenes small bowel, colon, GIT, cornea, lung, uterous or cervix in pregnant women Methylobacterium hematological, peritoneum, skin, soft tissue, bone spp. Mycobacterium lung/bronchi/trachea, lymph nodes pulmonary hilum, avium prostate, pancreas, spleen, skin, neck lymph nodes, esophagus, bone, hematological Mycobacterium colon, small bowel, joints bovis Mycobacterium lung/bronchi/trachea, lymph nodes pulmonary hilum, kansasii prostate, bone Mycobacterium skin, soft tissues, testes, eye leprae Mycobacterium skin, soft tissue, bone marinum Mycobacterium head and neck lymph nodes scrofulaceum Mycobacterium lung/bronchi/trachea, lymph nodes pulmonary hilum, tuberculosis prostate, peritoneum, pancreas, spleen, lymph nodes, small bowel, meninges, brain, spinal cord, kidney, ureter, bladder, muscle, esophagus, colon, testes, eye, ovaries, cervix, vagina, uterus, mediastinum, larynx, skin, hematological, pleura, joints Mycobacterium skin, soft tissue ulcerans other lung/bronchi/trachea, lymph nodes pulmonary hilum, skin, Mycobacterium soft tissues, bone, head and neck lymph nodes, joints spp. Myroides spp. kidney, bladder, ureter, skin, soft tissue, hematological Neisseria nasopharyx, oral, tonsil, prostate, penis, vagina, cervix, gonorrhoeae uterus, ovary/adnexae, peritoneum, skin, muscle, bone, liver, hematological, head and neck and inguinal and intra- abdominal lymph nodes, anus, joints Neorickettsia hematological, bone, lymph nodes, liver, spleen sennetsu Nocardia spp. lung/bronchi/trachea, pancreas, meninges, spinal cord, brain, skin, soft tissue, eye, bone, kidney, heart, hematological Orientia meninges, brain, spinal cord, hematological, skin, inguinal tsutsugamushi and axillary lymph nodes, spleen, lung/bronchi/trachea Pandoraea spp. lung/trachea/bronchi, hematological Pasteurella canis skin, soft tissue, hematological Pasteurella skin, soft tissue, hematological dagmatis Pasteurella skin, soft tissue, hematological stomatis Pediococcus spp. hematological, liver, colon Pityrosporum skin ovale Plesiomonas small bowel, colon, hematological, meninges, bone, gall shigelloides bladder, skin, soft tissue Pseudomonas lung/trachea/bronchi, hemaotogical, skin, soft tissue, bone, aeruginosa meninges, brain, eye, kidney, bladder, ureter, heart, joints other skin, soft tissue, lung/trachea/bronchi, mediastinum, Pseudomonas hematological spp. Ralstonia spp. hematological, meninges, bone Rhizobium spp. hematological, peritoneum, eye, kidney, bladder, ureter Rhodococcus lung/trachea/bronchi, hematological, brain, skin, lymph spp. nodes, bone, mediastinum, liver, spleen, soft tissue, spinal cord, meninges Rickettsia akari skin Rickettsia conorii lung/bronchi/trachea, lymph nodes pulmonary hilum, meninges, brain, spinal cord, hematolofical, skin, kidney, liver, spleen, pancreas Rickettsia felis skin, brain, spinal cord Rickettsia meninges, brain, spinal cord, hematological, prowazekii lung/bronchi/trachea, skin, spleen Rickettsia lung/bronchi/trachea, lymph nodes pulmonary hilum, rickettsiae meninges, brain, spinal cord, hematological, muscle, small bowel, liver, skin Rickettsia slovaca skin, head and neck lymph nodes Rickettsia typhi meninges, hematological, liver, kidney, brain, lung/bronchi/trachea, spleen Roseomonas spp. hematological, peritoneum, skin, soft tissue, bladder, kidney, ureter Salmonella spp. lung/bronchi/trachea, pancreas, spleen, intra-abdominal lymph nodes, stomach, small bowel, colon, meninges, skin, muscle, bone, hematological, heart, joints Shewanella spp. skin, soft tissue, eye, bone, hematological, peritoneum Shigella boydii colon Shigella colon dysenteriae Shigella flexneri colon Shigella sonnei colon Sphingobacterium brain, meninges, spinal cord, eye, skin, soft tissue spp. Sphingomonas hematological, meninges, peritoneum, skin, soft tissue, spp. kidney, bladder, ureter Spirillum minus skin, axillary/inguinal/neck lymph nodes, hematological, liver, spleen other Spirillum colon spp. Stenotrophomonas meninges, hematological, peritoneum, maltophilia lung/trachea/bronchi, eye, kidney, bladder, ureter, skin, soft tissue Streptobacillus skin, bone, hematological, lung/trachea/bronchi, meninges, moniliformis brain, liver, spleen Streptococcus skin, hematological, soft tissue iniae Streptococcus small bowel, nasopharynx, bone, meninges, zooepidemicus hematological, head and neck lymph nodes Streptomices spp. skin, soft tissue, lung/trachea/bronchi, mediastinum, brain, spinal cord, hematological, meninges, joints Treponema nasopharynx, tonsil, oral, meninges, brain, spinal cord, pallidum penis, vulva, vagina, anus, cervix, eye, hematological, inguinal and head and neck lymph nodes Tropheryma brain, spinal cord, hematological, small bowel, colon, whipplei heart, lung/trachea/bronchi, eye Tsukamurella skin, soft tissue, lung/trachea/bronchi, mediastinum, brain, spp. spinal cord, hematological, meninges Vibrio cholerae colon, small bowel Vibrio hematological, meninges cincinnatiensis Vibrio damsela skin, soft tissue Vibrio fluvialis small bowel, colon Vibrio furnissii small bowel, colon Vibrio hollisae small bowel, colon, skin, soft tissue Vibrio hematological metschnikovii Vibrio colon, small bowel parahaemolyticus Vibrio vulnificus soft tissue, blood, skin Yersinia nasopharynx, tonsil, small bowel, intra-abdominal lymph enterocolitica nodes, colon, muscle, lung/trachea/bronchi, liver, spleen, hematological, joints Yersinia pestis lung/trachea/bronchi, lymph nodes pulmonary hilum, inguinal/axillary/neck lymph nodes, oral, tonsil, hematological, skin, joints Yersinia small bowel, colon, abdominal lymph nodes, joints pseudotuberculosis

In some embodiments, microbial pathogens for use in the invention may be viral pathogens. Table 9 provides an exemplary list of viral pathogens together with the tissue and organ sites for which each viral species is understood to be a pathogen. Accordingly, one aspect of the invention involves utilizing immunogenic compositions that are specific for the named viruses to treat pathologies associated with infections caused by heterologous micro-organisms in the organs or tissues that are identified adjacent to the name of the virus in Table 9. For example, an antigenic composition derived from, or specific for, a vaccinia virus, may be used to treat a condition characterized by an infection by a heterologous micro-organism in the skin, hematological tissues, lymph nodes, brain, spinal cord, eye or heart.

TABLE 9 Viral Human Pathogens and Their Sites of Infection virus tissue/organ sites Vaccinia skin, hematological, lymph nodes, brain, spinal cord, eye, heart Variola (smallpox) skin, hematological, lymph nodes, brain Monkeypox skin, hematological, head and neck lymph nodes, brain, eye, lung/trachea/bronchi, pulmonary hilar lymph nodes, mediastinum, nasopharynx Cowpox skin, hematological, lymph nodes Parapoxviruses Skin Molluscum skin contagiosum Tanapox skin, hematological, axillary and inguinal lymph nodes Herpes Simplex nasopharynx, oral, tonsil, hematological, virus (1 and 2) lung/bronchi/trachea, pancreas, meninges, brain, spinal cord, inguinal and head/neck lymph nodes, penis, vulva, perineum, esophagus, liver, eye, skin, rectum, tonsil, mediastinum, anus, vagina, cervix Varicella-zoster nasopharynx, sinus, lung/trachea/bronchi, pulmonary hilar lymph nodes, hematological, pancreas, meninges, brain, spinal cord, esophagus, liver, eye, skin, heart, mediastinum Cytomegalovirus nasopharynx, lymph nodes, tonsil, hematological, lung/trachea/bronchi, pancreas, abdominal lymph nodes, brain, spinal cord, esophagus, small bowel, colon/rectum, eye, liver, heart, skin, mediastinum, esophagus Epstein-Barr virus nasopharynx, tonsil, oral, lymph nodes, hematological, lung, abdominal lymph nodes, brain, spinal cord, muscles, esophagus, liver, heart, skin, spleen, kidney, muscle, heart, lung/trachea/bronchi, pulmonary hilar lymph nodes, mediastinum Human herpesvirus 6 skin, hematological, lung/trachea/bronchi, pulmonary hilar lymph nodes, brain, meninges, liver Human herpesvirus 7 skin, brain, liver Human herpesvirus 8 nasopharynx, tonsil, hematological, skin, spleen, head and neck lymph nodes Simian herpes B brain, spinal cord, skin, hematological, lymph nodes virus Adenovirus nasopharynx, oral, larynx, trachea, bronchi, lung, lymph nodes, meninges, brain, spinal cord, small bowel, colon, liver, intra-abdominal lymph nodes, mediastinum, bladder, sinus, hematological, ureter, kidney, bladder, thyroid, heart BK virus kidney Human skin, anus, penis, vulva, cervix, vagina, oral papillomavirus Hepatitis B virus liver, pancreas, hematological, bone, joints Hepatitis D virus liver Parvovirus B19 skin, hematological, nasopharynx, bone, kidney, heart, liver, brain, meninges, joints Orthoreoviruses nasopharynx, small bowel, colon, oral, sinus, lymph nodes, skin, lung/trachea/bronchi, meninges, brain, spinal cord, liver Orbiviruses brain, muscle, hematological, Coltiviruses hematological, skin, muscle, oral, spleen, lymph nodes, meninges, brain Rotaviruses small bowel, colon, liver, hematological, pancreas, nasopharynx, billiary tract, meninges, brain Alphaviruses brain, spinal cord, small bowel, colon, hematological, skin, bone Rubella skin, hematological, head and neck lymph nodes, spleen, nasopharynx, bone, brain, tonsil, bronchi, liver, heart, joints Yellow fever virus hematological, liver, lung/trachea/bronchi, kidney, adrenal gland, spleen, lymph nodes, stomach, kidney Dengue fever virus hematological, lymph nodes, skin, spleen, muscle, liver, brain, nasopharynx, joints Japanese brain, hematological, spinal cord encephalitis virus West Nile brain, hematological, spinal cord, muscle, lymph nodes, encephalitis virus liver, spleen, pancreas, meninges St. Louis brain, hematological, spinal cord, meninges, muscle, encephalitis virus nasopharynx Tick-borne brain, hematological, spinal cord, muscle, meninges encephalitis virus other Flaviviruses hematological, brain, meninges, bone, muscles, skin, lymph nodes Hepatitis C virus hematological, liver, joints Hepatitis G virus liver Coronaviruses nasopharynx, sinus, oral, tonsil, larynx, lung/trachea/bronchi, pulmonary hilar lymph nodes, small bowel, colon, tonsil, hematological Toroviruses small bowel, colon, hematological Parainfluenza nasopharynx, sinus, tonsil, oral, larynx, viruses lung/trachea/bronchi, pulmonary hilar lymph nodes, meninges, hematological, mediastinum Mumps virus salivary glands, pancreas, brain, spinal cord, liver, testes, hematological, meninges, ovaries, bone, heart, kidney, thyroid, prostate, breast, joints Respiratory syncytial nasopharynx, tonsil, sinus, lung/trachea/bronchi, virus pulmonary hilar lymph nodes, mediastinum, hematological, oral, pleura Human nasopharynx, lung/trachea/bronchi, pulmonary hilar metapneumovirus lymph nodes, tonsil, sinus, mediastinum, hematological, oral, pleura, larynx, eye, skin, small bowel, colon Rubeola nasopharynx, sinus, hematological, lung/trachea/bronchi, pulmonary hilar lymph nodes, intra-abdominal lymph nodes, meninges, brain, spinal cord, liver, spleen, lymph nodes, skin, thymus, eye, oral, heart Hendra virus brain, meninges, lung/trachea/bronchi, kidney, hematological, muscle, Nipah virus brain, meninges, spleen, lymph nodes, thymus, lung/trachea/bronchi, kidneys, brain, spinal cord, meninges, hematological Vesicular stomatitis hematological, muscle, oral, tonsil, nasopharyngeal, virus lymph nodes, small bowel, colon Rabies virus skin, meninges, brain, spinal cord, oral, nasopharynx, salivary glands, hematological Lyssaviruses brain, spinal cord Influenza virus nasopharynx, laryngx, lung/trachea/bronchi, pulmonary hilar lymph nodes, meninges, muscle, hematological, mediastinum, muscle, sinus, tonsil, oral, eye, pleura, brain, spinal cord, salivary glands, thyroid, heart California hematological, brain, meninges encephalitis virus Hantaviruses hematological, kidney, eye, skin, oral, muscle, lung/trachea/bronchi other Bunyaviruses brain, hematological, muscle, meninges, spinal cord Lymphocytic hematological, muscle, lymph nodes, skin, brain, choriomeningitis meninges, testes, bone virus Lassa virus nasopharynx, brain, spinal cord, lung/trachea/bronchi, pulmonary hilar lymph nodes, mediastinum, muscle, testes, eye, heart, Machupo virus brain, meninges, hematological, muscle, eye, skin, lymph nodes, nasopharynx, small bowel, colon Junin virus brain, meninges, hematological, muscle, eye, skin, lymph nodes, nasopharynx, small bowel, colon Human T-Cell hematological, skin, lymph nodes, muscle, eye, bone, Lymphotropic lung, pulmonary hilar lymph nodes, spinal cord, brain viruses Poliovirus nasopharynx, lung/trachea/bronchi, pulmonary hilar lymph nodes, small bowel, neck and intra-abdominal lymph nodes, colon, hematological, liver, spleen, skin, brain, spinal cord, meninges, heart Coxsackieviruses nasopharynx, larynx, oral, tonsil, lung/trachea/bronchi, pulmonary hilar lymph nodes, mediastinum, pancreas, muscle, brain, meninges, small bowel, neck and intra- abdominal lymph nodes, colon, hematological, spleen, skin, eye, sinus, liver, testes, bone, pleura, salivary glands, heart Echoviruses nasopharynx, oral, tonsil, lung/trachea/bronchi, pulmonary hilar lymph nodes, muscle, brain, meninges, small bowel, neck and intra-abdominal lymph nodes, colon, hematological, mediastinum, spleen, skin, eye, sinus, liver, pancreas, testes, bone, salivary glands, heart other Enteroviruses lung/trachea/bronchi, pulmonary hilar lymph nodes, meninges, brain, skin, heart, joints Hepatitis A virus small bowel, colon, hematological, liver, spleen, brain, spinal cord, gallbladder, pancreas, kidney Rhinoviruses nasopharynx, sinus, oral, tonsil, larynx, lung/trachea/bronchi, pulmonary hilar lymph nodes Noroviruses and small bowel, colon other Caliciviruses Astroviruses small bowel, colon Picobirnaviruses small bowel, colon Hepatitis E virus liver, small bowel, colon, hematological

The cumulative information in Tables 6 through 9 provides an extensive identification of microbial pathogens that may be used in the formulation of antigenic compositions of the invention, together with an identification of the tissues or organs in which these organisms are pathogenic, and accordingly identifies the correspondence between selected tissues or organs in which an infection by a heterologous organism is situated, and the organisms that may be used to produce antigenic formulations for treating the condition.

In some embodiments, the microbial pathogen selected for use in antigenic compositions of the invention may be one that is a common cause of acute infection in the tissue or organ in which the heterologous infection is to be treated. Table 10 identifies bacterial and viral pathogens of this kind, together with the tissues and organs in which they commonly cause infection. Accordingly, in selected embodiments, an infection residing in a tissue identified in the first column of Table 10 may be treated with an antigenic composition that comprises antigenic determinants for one or more of the heterologous pathogenic organisms listed in the second column of Table 10. For example, an infection in the skin may be treated with an antigenic composition comprising antigenic determinants of one or more of the following heterologous organisms: Staphylococcus aureus, Beta hemolytic streptococci group A, B, C and G, Corynebacterium diptheriae, Corynebacterium ulcerans, Pseudomonas aeruginosa, rubeola, rubella, varicella-zoster, echoviruses, coxsackieviruses, adenovirus, vaccinia, herpes simplex, or parvo B19.

TABLE 10 Common Causes of Acute Infection (Bacterial and Viruses) For Each Tissue/Organ Site Tissue/ Common Bacterial or Viral Pathogens of specific organ site tissue/organ site Skin Staphylococcus aureus, Beta hemolytic streptococci group A, B, C and G, Corynebacterium diptheriae, Corynebacterium ulcerans, Pseudomonas aeruginosa rubeola, rubella, varicella-zoster, echoviruses, coxsackieviruses, adenovirus, vaccinia, herpes simplex, parvo B19 Soft tissue (i.e. Streptococcus pyogenes, Staphylococcus aureus, fat and muscle) Clostridium perfringens, other Clostridium spp. (e.g., sarcoma) influenza, coxsackieviruses Breast Staphylococcus aureus, Streptococcus pyogenes Lymph nodes: Staphylococcus aureus, Streptococcus pyogenes head and neck Epstein-Barr, cytomegalovirus, adenovirus, measles, rubella, herpes simplex, coxsackieviruses, varicella-zoster Lymph nodes: Staphylococcus aureus, Streptococcus pyogenes axillae/arm measles, rubella, Epstein-Barr, cytomegalovirus, adenovirus, varicella-zoster Lymph nodes: viridans streptococci, Peptococcus spp., mediastinal Peptostreptococcus spp., Bacteroides spp., Fusobacterium spp., Mycobacterium tuberculosis measles, rubella, Epstein-Barr, cytomegalovirus, varicella- zoster, adenovirus Lymph nodes: Streptococcus pneumoniae, Moraxella catarrhalis, pulmonary Mycoplasma pneumoniae, Klebsiella pneumoniae, hilum Haemophilus influenza, Chlamydophila pneumoniae, Bordetella pertussis, Mycobacterium tuberculosis influenza, adenovirus, rhinovirus, coronavirus, parainfluenza, respiratory syncytial virus, human metapneumovirus, coxsackievirus Lymph nodes: Yersinia enterocolitica, Yersinia pseudotuberculosis, intra-abdominal Salmonella spp., Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis measles, rubella, Epstein-Barr, cytomegalovirus, varicella- zoster, adenovirus, influenza, coxsackieviruses Lymph nodes: Staphylococcus aureus, Streptococcus pyogenes inguinal/leg measles, rubella, Epstein-Barr, cytomegalovirus, herpes simplex Hematological Staphylococcus aureus, Streptococcus pyogenes, (e.g. leukemias, coagulase-negative staphylococci, Enterococcus spp., multiple myeloma) Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, Bacteroides fragilis, Streptococcus pneumoniae, group B streptococci rubeola, rubella, varicella-zoster, echoviruses, coxsackieviruses, adenovirus, Epstein-Barr, cytomegalovirus, herpes simplex Bone Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, other streptococci spp., Escherichia coli, Pseudomonas spp., Enterobacter spp., Proteus spp., Serratia spp. parvovirus B19, rubella, hepatitis B Joint Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, other streptococci spp., Escherichia coli, Pseudomonas spp., Enterobacter spp., Proteus spp., Serratia spp., Neisseria gonorrhea, salmonella species, Mycobacterim tuberculosis, Hemophilus influenza parvovirus B19, rubella, hepatitis B Scedosporium prolificans Meninges Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, Listeria monocytogenes echoviruses, coxsackieviruses, other enteroviruses, mumps Brain Streptococcus spp. (including S. anginosus, S. constellatus, S. intermedius), Staphylococcus aureus, Bacteroides spp., Prevotella spp., Proteus spp., Escherichia coli, Klebsiella spp., Pseudomonas spp., Enterobacter spp., Borrelia burgdorferi coxsackieviruses, echoviruses, poliovirus, other enteroviruses, mumps, herpes simplex, varicella-zoster, flaviviruses, bunyaviruses Spinal cord Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, Listeria monocytogenes, Borrelia burgdorferi coxsackieviruses, echoviruses, poliovirus, other enteroviruses, mumps, herpes simplex, varicella-zoster, flaviviruses, bunyaviruses Eye/Orbit Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus milleri, Escherichia coli, Bacillus cereus, Chlamydia trachomatis, Haemophilus influenza, Pseudomonas spp., Klebsiella spp., Treponema pallidum adenoviruses, herpes simplex, varicella-zoster, cytomegalovirus Salivary glands Staphylococcus aureus, viridans streptococci (e.g., Streptococcus salivarius, Streptococcus sanguis, Streptococcus mutans), Peptostreptococcus spp., Bacteroides spp., and other oral anaerobes mumps, influenza, enteroviruses, rabies Oral Prevotella melaninogenicus, anaerobic streptococci, viridans streptococci, Actinomyces spp., Peptostreptococcus spp., Bacteroides spp., and other oral anaerobes herpes simplex, coxsackieviruses, Epstein-Barr Tonsil Streptococcus pyogenes, Group C and G B-hemolytic streptococci rhinoviruses, influenza, coronavirus, adenovirus, parainfluenza, respiratory syncytial virus, herpes simplex Sinus Streptococcus pneumoniae, Haemophilus influenza, Moraxella catarrhalis, α-streptococci, anaerobic bacteria (e.g., Prevotella spp.), Staphylococcus aureus rhinoviruses, influenza, adenovirus, parainfluenza Nasopharynx Streptococcus pyogenes, Group C and G B-hemolytic streptococci rhinoviruses, influenza, coronavirus, adenovirus, parainfluenza, respiratory syncytial virus, herpes simplex Thyroid Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae mumps, influenza Larynx Mycoplasma pneumoniae, Chlamydophila pneumoniae, Streptococcus pyogenes rhinovirus, influenza, parainfluenza, adenovirus, coronavirus, human metapneumovirus Trachea Mycoplasma pneumoniae parainfluenza, influenza, respiratory syncytial virus, adenovirus Bronchi Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bordetella pertussis, Streptococcus pneumoniae, Haemophilus influenzae influenza, adenovirus, rhinovirus, coronavirus, parainfluenza, respiratory syncytial virus, human metapneumovirus, coxsackievirus Lung Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Klebsiella pneumoniae, Haemophilus influenza influenza, adenovirus, respiratory syncytial virus, parainfluenza Pleura Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Bacteroides fragilis, Prevotella spp., Fusobacterium nucleatum, peptostreptococcus spp., Mycobacterium tuberculosis influenza, adenovirus, respiratory syncytial virus, parainfluenza Mediastinum viridans streptococci, Peptococcus spp., Peptostreptococcus spp., Bacteroides spp., Fusobacterium spp. measles, rubella, Epstein-Barr, cytomegalovirus Heart Streptococcus spp. (including S. mitior, S. bovis, S. sanguis, S. mutans, S. anginosus), Enterococcus spp., Staphylococcus spp., Corynebacterium diptheriae, Clostridium perfringens, Neisseria meningitidis, Salmonella spp. enteroviruses, coxsackieviruses, echoviruses, poliovirus, adenovirus, mumps, rubeola, influenza Esophagus Actinomyces spp., Mycobacterium avium, Mycobacterium tuberculosis, Streptococcus spp. cytomegalovirus, herpes simplex, varicella-zoster Stomach Streptococcus pyogenes, Helicobacter pylori cytomegalovirus, herpes simplex, Epstein-Barr, rotaviruses, noroviruses, adenoviruses Small bowel Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, cytomegalovirus Colon/Rectum Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri adenoviruses, astroviruses, caliciviruses, noroviruses, rotaviruses, cytomegalovirus Anus Streptococcus pyogenes, Bacteroides spp., Fusobacterium spp., anaerobic streptococci, Clostridium spp., E. coli, Enterobacter spp., Pseudomonas aeruginosa, Treponema pallidum herpes simplex Perineum Escherichia coli, Klebsiella spp., Enterococcus spp., Bacteroides spp., Fusobacterium spp., Clostridium spp., Pseudomonas aeruginosa, anaerobic streptococci, Clostridium spp., E. coli, Enterobacter spp. herpes simplex Liver Escherichia coli, Klebsiella spp., Streptococcus (anginosus group), Enterococcus spp., other viridans streptococci, Bacteroides spp. hepatitis A, Epstein-Barr, herpes simplex, mumps, rubella, rubeola, varicella-zoster, coxsackieviruses, adenovirus Gallbladder Escherichia coli, Klebsiella spp., Enterobacter spp., enterococci, Bacteroides spp., Fusobacterium spp., Clostridium spp., Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri Biliary tract Escherichia coli, Klebsiella spp., Enterobacter spp., Enterococci spp., Bacteroides spp., Fusobacterium spp., Clostridium spp., Salmonella enteriditis, Yersinia enterocolitica, Shigella flexneri hepatitis A, Epstein-Barr, herpes simplex, mumps, rubella, rubeola, varicella-zoster, cocsackieviruses, adenovirus Pancreas Escherichia coli, Klebsiella spp., Enterococcus spp., Pseudomonas spp., Staphylococcal spp., Mycoplasma spp., Salmonella typhi, Leptospirosis spp., Legionella spp. mumps, coxsackievirus, hepatitis B, cytomegalovirus, herpes simplex 2, varicella-zoster Spleen Streptococcus spp., Staphylococcus spp., Salmonella spp., Pseudomonas spp., Escherichia coli, Enterococcus spp. Epstein-Barr, cytomegalovirus, adenovirus, measles, rubella, coxsackieviruses, varicella-zoster Adrenal gland Streptococcus spp., Staphylococcus spp., Salmonella spp., Pseudomonas spp., Escherichia coli, Enterococcus spp. varicella-zoster Kidney Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providentia spp., Morganella spp., Enterococcus faecalis, Pseudomonas aeruginosa BK virus, mumps Ureter Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providentia spp., Morganella spp., Enterococcus spp. Bladder Escherichia coli, Proteus mirabilis, Proteus vulgatus, Providentia spp., Morganella spp., Enterococcus faecalis, Corynebacterium jekeum adenovirus, cytomegalovirus Peritoneum Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumonia, Escherichia coli, Klebsiella spp., Proteus spp., Enterococci spp., Bacteroides fragilis, Prevotella melaninogenica, Peptococcus spp., Peptostreptococcus spp., Fusobacterium spp., Clostridium spp. Retroperitoneal Escherichia coli, Staphylococcus aureus area Prostate Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus mirabilis, Enterococci spp., Pseudomonas spp., Corynebacterium spp., Neisseria gonorrhoeae herpes simplex Testicle Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus spp., Streptococcus spp., Salmonella enteriditis mumps, coxsackievirus, lymphocytic choriomeningitis virus Penis Staphylococcus aureus, Streptococcus pyogenes, Neisseria gonorrhoeae, Treponema pallidum herpes simplex, human papillomavirus Ovary/Adnexae Neisseria gonorrhoeae, Chlamydia trachomatis, Gardenerella vaginalis, Prevotella spp., Bacteroides spp., Peptococcus spp. Streptococcus spp., Escherichia coli Uterus Neisseria gonorrhoeae, Chlamydia trachomatis, Gardenerella vaginalis, Prevotella spp., Bacteroides spp., Peptococcus spp., Streptococcus spp., Escherichia coli Cervix Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum herpes simplex Vagina Gardenerella vaginalis, Prevotella spp., Bacteroides spp., peptococci spp., Escherichia coli, Neisseria gonorrhoeae, Chlamydia Trachomatis, Treponema pallidum, herpes simplex Vulva Staphylococcus aureus, Streptococcus pyogenes, Treponema pallidum herpes simplex

In selected embodiments, particular microbial pathogens are suited for treatment of particular pathologies associated with heterologous microbal infections located in the tissue or organ within which the organism is pathogenic, examples of selected embodiments are set out in Table 10. These are exemplary embodiments, and not an exhaustive list of the alternative formulations for use in accordance with the invention.

The specific microbes which commonly cause infection in a specific tissue or organ may vary by geographical location. For example, Mycobacterium tuberculosis is a more common cause of lung infection in some geographical locations and populations than in others and therefore, while M. tuberculosis may not be a common lung pathogen in some geographic and population groups it may be a common lung pathogen in others. Table 10 is thus not an exhaustive list of common pathogens for all geographic locations and population groups. It is understood that a clinical microbiologist skilled in the art could determine the common pathogenic species in a particular geographic area or population group for a specific tissue or organ site in accordance with the invention. For veterinary use, there will of course be specific pathogens that are common in selected tissues of selected species, and this may also vary geographically.

In selected embodiments, the invention involves diagnostic steps to assess a patient's previous exposure to microbial pathogens. For example, the diagnostic steps may include taking a medical history of exposure to selected pathogens, and/or evaluating a patient's immune response to a selected pathogen. For example, a serology test may be conducted to detect antibodies to selected pathogens in a patient's sera. In connection with this aspect of the invention, antigenic determinants of a selected microbial pathogen may be chosen for use in an immunogenic composition on a selected patient based on a diagnostic indication that the patient has had one or more prior exposure(s) to the pathogen, for example by virtue of the presence of antibodies to antigenic determinants of that pathogen in the patient's sera.

In further selected embodiments, the invention involves diagnostic steps to assess a patient's immunological response to treatment with a selected immunogenic composition. For example, the diagnostic steps may include evaluating a patient's immune response to the antigenic determinants of that immunogenic composition, for example using a serological test to detect antibodies to those antigenic determinants. In connection with this aspect of the invention a treatment with a selected immunogenic composition may be continued if the evaluation indicates that there is an active immunological response to the antigenic determinants of that composition, and the treatment may be discontinued, and an alternative treatment with a different immunogenic composition may be initiated, if the evaluation indicates that there is not a sufficiently active immunological response to the antigenic determinants of the immunogenic composition.

In selected embodiments, the microbial pathogen selected for use in antigenic compositions of the invention may be one that is the most common cause of acute infection in the tissue or organ in which the heterologous infection is to be treated. For example, for the treatment of pathologies associated with infections of the bone, Staphylococcus aureus would be the bacterial species selected for treatment of infections caused by heterologous organisms; for the treatment of infections in lung tissue, Streptococcus pneumoniae would be selected for treatment of infections caused by heterologous organisms; for the treatment of breast infections, Staphylococcus aureus would be selected for treatment of infections caused by heterologous organisms; for the treatment of kidney or bladder infections, Escherichia coli would be selected for treatment of infections caused by heterologous organisms; and for the treatment of infections in the colon, Escherichia coli would be the bacterial species selected for treatment of infections caused by heterologous organisms. It is understood that a clinical microbiologist skilled in the art could determine the most frequently pathogenic species, bacterial or viral, for each specific tissue or organ in accordance with the invention. In selected embodiments, only antigenic determinants of the most common pathogen for the particular tissue or organ are used to treat heterologous infections of that tissue or organ. In alternative embodiments, antigenic determinants of the most common pathogen for the particular tissue or organ could be used in combination with antigenic determinants of other pathogens that are known to be pathogenic in the of that particular tissue or organ, preferentially selecting from the more common pathogens.

In some embodiments, the invention provides antigenic compositions in which a threshold proportion of antigenic determinants selected in accordance with the invention are used, relative to any other antigenic determinants in the composition. For example, antigenic compositions may have greater than X % of the antigenic determinants therein derived from pathogenic (or commonly pathogenic, or most commonly pathogenic) species, where X may for example be 10, 30, 40, 50, 60, 70, 80, 90, 95 or 100 (or any integer value between 10 and 100). For example, at least X % of the antigenic determinants in the antigenic composition may be specific for microbial pathogens that are pathogenic (or commonly pathogenic or most commonly pathogenic) in the specific organ or tissue of the patient within which the heterologous infection is situated. Using an alternative measure, of the total number of microbial pathogens in the antigenic composition, at least X % may be selected to be microbial pathogens that are pathogenic (or commonly pathogenic or most commonly pathogenic) in the specific organ or tissue of the patient within which the heterologous microbial infection is situated. In some embodiments, the antigenic composition may accordingly consist essentially of antigenic determinants of one or more microbial pathogens that are each pathogenic (or commonly pathogenic or most commonly pathogenic) in the specific organ or tissue of the patient within which the heterologous infection is situated.

In some embodiments, the invention comprises the use of bacterial or viral vaccines or formulations that are approved for other purposes (e.g., poliomyelitis vaccine, H. influenza vaccine, meningococcal vaccine, pneumococcal vaccine, influenza vaccine, hepatitis B vaccine, hepatitis A vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, measles vaccine, mumps vaccine, rubella vaccine, varicella vaccine, BCG vaccine, cholera vaccine, Japanese encephalitis vaccine, rabies vaccine, typhoid vaccine, yellow fever vaccine, small pox vaccine, etc.) for use as treatments of infections caused by heterologous micro-organisms by selecting a vaccine containing a pathogen (or antigenic constituent of a pathogen) that is pathogenic in the specific organ or tissue of the patient within which the heterologous infection is situated by consulting Tables 6-10. For example, a S. pneumoniae vaccine, either a whole cell vaccine or a vaccine comprised of one or more antigenic components of S. pneumoniae (e.g., pneumococcal polysaccharide-23-valent) could be used to treat a heterologous infection at any of the following sites in which S. pneumoniae is listed as a common pathogen in Table 10: pulmonary hilar lymph nodes, bone, meninges, spinal cord, eye/orbit, sinus, thyroid, bronchi, lungs, pleura or peritoneum. As a further example, a hepatitis B vaccine could be used to treat a heterologous infection at any of the following sites in which hepatitis B virus is listed as a pathogen in Table 9, as follows: liver, pancreas, or hematological infections.

In some embodiments, selected compositions and methods are specifically excluded from the scope of the invention. For example, the use of the a formulation of antigens of a particular microbial pathogen in the treatment of a pathology associated with infection by that organism. For example, selected embodiments exclude the use of PVF or MRV vaccines for the treatment of lung infections caused by the organisms that are present in those formulations.

Example 1: Murine Studies Example 1a: Illustrating the Influence of a Heat Inactivated Klebsiella pneumoniae Antigenic Composition on Monocyte/Macrophage and Dendritic Cell Populations in Mice

The following methods and materials were utilized in this Example:

Mice.

C57BL/6 female mice 7-8 weeks of age were ordered from Harlan Labs (Livermore, Calif.) for these studies.

Antibodies and Reagents.

The following antibodies were used in this Example: anti-I-A/I-E FITC (MHC Class II; M5/114.15.2); anti-Gr-1 PE (RB6-8C5); anti-CD11b PerCP-Cy5 (M1/70); anti-CD11c APC (N418); anti-CD4 FITC (GK1.5); anti-NK1.1 PE (PK136); anti-CD8a eFluor780 (53-6.7); anti-CD44 APC (IM7). All antibodies were acquired from eBioscience (San Diego, Calif.). Liberase TM and DNAse I was acquired from Roche. All media was from HyClone (Fisher).

Treatment with Antigenic Compositions.

Heat killed K. pneumoniae with phenol (KO12 [5.0 OD600 units]) was diluted 1/10 in PBS containing 0.4% phenol and 100 μl was injected subcutaneously on day 0, 2, 4, and 6 into 4 mice. Control mice (n=5) were injected on day 0, 2, 4, and 6 with PBS.

Brochoalveolar Lavage.

On day 7 mice were sacrificed and a bronchoalveolar lavage (BAL) was performed by exposing the trachea followed by insertion of a 22G catheter attached to a 1 ml syringe. 1 ml of PBS was injected into the lungs and removed and placed into a 1.5 ml microcentrifuge tube. The lungs were subsequently washed 3 more times with 1 ml of PBS and the fluid was pooled. The first wash from each mouse was centrifuged at 400×g and the supernatant was frozen for cytokine analysis. The final 3 ml of lavage fluid was centrifuged and the cells were pooled with the cell pellet from the first lavage. The cells were counted and stained with antibodies specific for MHC class 11, Ly6G/C, CD11b, and CD11c. After staining the cells were washed and analyzed on a FACS Calibur flow cytometer.

Lung Digestion.

After BAL was performed the lungs were placed in 5 ml of RPMI containing 417.5 μg/ml Liberase TL (Roche) and 200 μg/ml DNAse I (Roche). The lungs were then digested at 37° C. for 30 mins. After digestion the lungs were forced through a 70 um cell strainer to create a single cell suspension. The cells were then centrifuged, washed, resuspended in FACS Buffer (PBS with 2% FCS and 5 mM EDTA) and counted. After counting the cells were stained and analyzed by FACS using the same antibodies as for the BAL cells.

Peritoneal Lavage.

1 ml of PBS was injected into the peritoneum of mice using a 1 ml syringe attached to a 25G needle after BAL. The abdomen was massaged for 1 minute and 0.5 ml of PBS was recovered from the peritoneum using a 1 ml pipet. The lavage fluid was put in a 1.5 ml centrifuge tube, centrifuged at 400×g for 5 mins, and resuspended in FACS buffer prior to staining and FACS analysis.

Spleen and Lymph Node Analysis.

The spleen and draining lymph node were removed after BAL and peritoneal lavage and placed in PBS. The spleen was disrupted by mashing through a 70 μm cell strainer (Fisher) and the lymph node was disrupted using the rubber end of the plunger from a 1 ml syringe. After disruption, the single cell suspension from the spleen and lymph nodes was centrifuged, washed once with FACS Buffer, and resuspended in FACS Buffer prior to counting, staining, and FACS analysis.

FACS Analysis.

Cells were stained on ice for 20 mins in 96 well plates using 50 ul of antibodies diluted in FACS buffer. After 20 mins, 100 μl of FACs buffer was added to the wells and the plates were centrifuged at 400×g for 5 mins. Subsequently the media was removed and the cells were washed 1 more time with FACS buffer. After the final wash the cells were resuspended in 200 μl of FACS buffer and the data was acquired using a FACS Calibur flow cytometer (BD). A minimum of 20,000 live events were collected for all samples except the BAL where a minimum of 5,000 events was collected.

The following results were obtained in this Example.

Normal mice were treated with a K. pneumoniae antigenic composition on day 0, 2, 4, and 6. On day 7 the mice were sacrificed and the bronchoalveolar lavage fluid, lung tissue, peritoneal lavage fluid, lymph nodes, and spleen was analyzed for changes in monocyte and macrophages. An increase in the number of acute inflammatory blood monocytes/macrophages, defined by high expression of CD11b and Gr-1 (same marker as Ly6c), and F4/80 in the lymph node draining the site of injection of the K. pneumoniae antigenic composition was observed (see: FIG. 1A). These acute inflammatory monocytes/macrophages also express very high levels of MHC class II molecules suggesting exposure to bacterial antigens. Importantly, treatment of mice with the K. pneumoniae antigenic composition for one week led to a marked increase in the frequency of acute inflammatory monocytes in the bronchoalveolar lavage fluid and in the lungs (i.e., the targeted organ) but not in the spleen or peritoneum of treated mice, suggesting that treatment can induce homing of monocytes specifically to the lungs without affecting other organs (see: FIG. 1B). Monocytes can differentiate into dendritic cells (DCs) in the lungs and consistent with our observations of a marked increase in monocyte recruitment it was also observed that there was a marked increase in the frequency of cells displaying markers for mature DCs (see: FIG. 1C).

As illustrated in FIGS. 1A to 1C, treatment with a K. pneumoniae antigenic composition for 7 days resulted in a marked increase (compared to treatment with placebo=PBS) in both acute inflammatory monocytes and dendritic cells in the lungs of mice. As illustrated in FIGS. 1A to 1C, mice were treated with either a K. pneumoniae antigenic composition for or PBS on day 0, 2, 4, and 6. On day 7, the mice were sacrificed and the total number of A) and B) inflammatory monocytes (CD11b+ Gr-1+ cells) and C) dendritic cells (CD11c+ MHC class II+ cells) were determined by flow cytometry in the lung and spleen. The error bars depicted in A) represent the mean of 4-5 mice per group.

Example 1B. Illustrating the Influence of a Heat Inactivated Klebsiella pneumoniae Antigenic Composition and a Heat Inactivated E. coli Antigenic Composition on Monocyte/Macrophage, Dendritic Cell, and Effector Cell Populations in Mice

The following methods and materials were utilized in this Example:

Mice.

C57BL/6 female mice 7-8 weeks of age were ordered from Harlan Labs (Livermore, Calif.) for these studies.

Antibodies and Reagents.

The following antibodies were used: anti-I-A/I-E FITC (MHC Class II; M5/114.15.2); anti-Gr-1 PE (RB6-8C5); anti-CD11b PerCP-Cy5 (M1/70); anti-CD11c APC (N418); anti-CD4 FITC (GK1.5); anti-NK1.1 PE (PK136); anti-CD8a eFluor780 (53-6.7); anti-CD44 APC (IM7). All antibodies were acquired from eBioscience (San Diego, Calif.). Liberase TM and DNAse I was acquired from Roche. All media was from HyClone (Fisher).

Treatment with Antigenic Compositions.

Heat-killed K. pneumoniae with phenol (K. pneumoniae; lot KO12; 5.0 OD600 units) was diluted 1/10 in PBS containing 0.4% phenol and 100 ul was injected subcutaneously on day 0, 2, 4, and 6 into 5 mice. Heat-killed E. coli (lot; 5.0 OD600 units) was diluted 1/10 in containing 0.4% phenol and 100 μl was injected subcutaneously on day 0, 2, 4, and 6 into 5 mice. Control mice (n=5) were injected on day 0, 2, 4, and 6 with PBS.

Brochoalveolar Lavage.

On day 7 mice were sacrificed and a bronchoalveolar lavage (BAL) was performed by exposing the trachea followed by insertion of a 22G catheter attached to a 1 ml syringe. 1 ml of PBS was injected into the lungs and removed and placed into a 1.5 ml microcentrifuge tube. The lungs were subsequently washed 3 more times with 1 ml of PBS and the fluid was pooled. The first wash from each mouse was centrifuged at 400×g and the supernatant was frozen for cytokine analysis. The final 3 ml of lavage fluid was centrifuged and the cells were pooled with the cell pellet from the first lavage. The cells were counted and stained with antibodies specific for MHC class II, Ly6G/C, CD11b, and CD11c. After staining the cells were washed and analyzed on a FACS Calibur flow cytometer.

Lung Digestion.

After BAL was performed the lungs were placed in 5 ml of RPMI containing 417.5 μg/ml Liberase TL (Roche) and 200 μg/ml DNAse I (Roche). The lungs were then digested at 37° C. for 30 mins. After digestion the lungs were forced through a 70 μm cell strainer to create a single cell suspension. The cells were then centrifuged, washed, resuspended in FACS Buffer (PBS with 2% FCS and 5 mM EDTA) and counted. After counting the cells were stained and analyzed by FACS using the same antibodies as for the BAL cells.

Peritoneal Lavage.

1 ml of PBS was injected into the peritoneum of mice using a 1 ml syringe attached to a 25G needle after BAL. The abdomen was massaged for 1 minute and 0.5 ml of PBS was recovered from the peritoneum using a 1 ml pipet. The lavage fluid was put in a 1.5 ml centrifuge tube, centrifuged at 400×g for 5 mins, and resuspended in FACS buffer prior to staining and FACS analysis.

Spleen and Lymph Node Analysis.

The spleen and draining lymph node were removed after BAL and peritoneal lavage and placed in PBS. The spleen was disrupted by mashing through a 70 μm cell strainer (Fisher) and the lymph node was disrupted using the rubber end of the plunger from a 1 ml syringe. After disruption, the single cell suspension from the spleen and lymph nodes was centrifuged, washed once with FACS Buffer, and resuspended in FACS Buffer prior to counting, staining, and FACS analysis.

FACS Analysis.

Cells were stained on ice for 20 mins in 96 well plates using 50 μl of antibodies diluted in FACS buffer. After 20 mins, 100 μl of FACs buffer was added to the wells and the plates were centrifuged at 400×g for 5 mins. Subsequently the media was removed and the cells were washed 1 more time with FACS buffer. After the final wash the cells were resuspended in 200 μl of FACS buffer and the data was acquired using a FACS Calibur flow cytometer (BD). A minimum of 20,000 live events were collected for all samples except the BAL where a minimum of 5,000 events was collected.

The following results were obtained in this Example:

As illustrated in FIG. 2, mice were treated on day 0, 2, 4, and 6 with either a K. pneumoniae antigenic composition, an E. coli antigenic composition or PBS. On day 7 the mice were sacrificed and the total number of inflammatory monocytes (CD11b+ Gr-1+ cells) and dendritic cells (CD11c+ MHC class II+ cells) were determined by flow cytometry in the peritoneal lavage fluid, lungs, lymph node and spleen. Error bars in FIG. 18 represent the standard deviation from 5 mice. *p-value<0.05 using a Student's t-test.

FIG. 2 illustrates that treatment with a K. pneumoniae antigenic composition, but not an E. coli antigenic composition treatment, markedly increased the number of monocytes and DCs in the lungs of mice. In contrast to the lungs, K. pneumoniae did not lead to an increase in monocytes in the peritoneum of the mice whereas E. coli did. Importantly, there was only a slight increase in the number of inflammatory monocytes and no increase in DCs in the spleens of mice treated with either K. pneumoniae or E. coli suggesting that the effects of the therapies are not general and are, in fact, specific for a particular organ site. In addition to looking at the effects of treatment on inflammatory monocytes and DCs in the lungs of mice, we also looked at changes in other leukocytes such as cytotoxic CD8 T cells, CD4 T helper cells, and natural killer (NK) cells.

FIG. 3 illustrates that a K. pneumoniae antigenic composition, but not PBS or an E. coli antigenic composition, resulted in a marked increase in the frequency and total numbers of NK cells, CD4 and CD8 T cells in the lungs of treated mice. This Example demonstrates that subcutaneous injection of a killed bacterial species which normally causes lung infection can promote the accumulation of leukocytes in the lungs without the presence of any inflammation in that site. In addition, it demonstrates that this effect is specific to the targeted site and that it is also specific to the bacterial constituents of the treatment used.

As illustrated in FIG. 3, mice were treated on day 0, 2, 4, and 6 with either a K. pneumoniae antigenic composition, an E. coli antigenic composition, or PBS. On day 7, the mice were sacrificed and the total number of CD4 T cells, CD8 T cells, and natural killer (NK) cells were determined by flow cytometry. Error bars represent the sd of values obtained from 5 mice per group. *p-value<0.05 using a Student's t-test.

Example 1C. Illustrating the Effects of Heat, Irradiation, and Phenol Inactivation on K. pneumoniae Antigenic Compositions, Including Leukocyte Recruitment into the Lungs of Mice, and the Effects of Phenol as a Preservative

The following methods and materials were utilized in this Example:

Mice.

C57BL/6 female mice 7-8 weeks of age were ordered from Harlan Labs (Livermore, Calif.) for these studies.

Antigenic Compositions.

Heat killed K. pneumoniae antigenic composition with phenol (KO12), heat killed K. pneumoniae antigenic composition without phenol (KO25), irradiated K. pneumoniae antigenic composition without phenol (KO24), and phenol killed K. pneumoniae antigenic composition without phenol (KO25) were used in this study. All bacterial formulations were at a concentration of 5.0 OD units in saline. For 1/10 dilution, 1 ml of bacterial formulation was added to 9 ml of DPBS and mixed immediately and then again prior to injection. For 1/100 dilution, 0.1 ml of bacterial formulation will be added to 9.9 ml of DPBS and mixed immediately and then again prior to injection. For dilutions of heat-killed Klebsiella pneumoniae antigenic composition with phenol, the dilutions were carried out as above using a DPBS solution containing 0.4% phenol (w/v). To prepare the 0.4% phenol in DPBS, first a 5% phenol solution was prepared by adding 0.5 g of solid phenol (Sigma Aldrich, St. Louis, Mo.) to 10 ml of DPBS (Hyclone, Logan, Utah) This solution was filtered through a 0.22 um filter (Millipore, Billerica, Mass.) and stored at 4° C. Immediately prior to use the 5% phenol solution was diluted 1 ml in 12.5 ml DPBS and used to prepare the bacterial formulations.

Treatment with Antigenic Compositions.

5 mice per group were treated subcutaneously on day 0, 2, 4, and 6 with 0.1 ml of a heat-killed K. pneumoniae antigenic composition diluted 1/10 in PBS or PBS with 0.4% phenol, 0.1 ml of an irradiated K. pneumoniae antigenic composition diluted 1/10 in PBS, or a phenol inactivated K. pneumoniae antigenic composition diluted 1/10 with PBS or PBS with 0.4% phenol. On day 7 the mice were sacrificed and leukocyte recruitment to the lungs was analyzed as in Example 1B.

The following results were obtained in this Example:

In this example, we used leukocyte recruitment to the lungs as a surrogate of efficacy to compare the efficacy of K. pneumoniae antigenic compositions inactivated by various methods. FIGS. 4A to 4B illustrate that, for both heat killed and phenol killed K. pneumoniae antigenic compositions, the addition of phenol (0.4%) as a preservative, increased efficacy, as measured by cellular recruitment. In some embodiments, a small amount of phenol (i.e., 0.4% as a preservative) may stabilize a component of the bacterial cell wall, for example a component that is important in antigen pattern recognition and activating an optimal targeted response. In comparing the 3 formulations containing phenol as a preservative (i.e., heat killed, phenol killed and radiation killed), irradiated K. pneumoniae antigenic composition led to the greatest recruitment of acute inflammatory monocytes, DCs, NK cells, and T cells to the lungs, followed by phenol killed K. pneumoniae antigenic composition, with heat killed K. pneumoniae antigenic composition resulting in the least cellular recruitment.

As illustrated in FIGS. 4A to 4B, mice were treated on day 0, 2, 4, and 6 with K. pneumoniae antigenic composition inactivated by heat (HKWP) or without (HKnp) phenol preservative, inactivated with phenol with (PKWP) or without (PKnp) phenol preservative, or K. pneumoniae antigenic composition inactivated by irradiation with phenol preservative (IRWP). On day 7 the mice were sacrificed and the total numbers of (A) inflammatory monocytes (CD11b+ Gr-1+) and DCs (CD11c+ Iab+) or (B) CD4 T cells, CD8 T cells, and natural killer (NK) cells were determined by flow cytometry. Error bars represent the sd of values from 5 mice per group. *p-value<0.05 compared to mice treated with IRWP using a Student's t-test.

Example 2: Site Specificity Studies

With a focus on investigating the M1/M2 phenotypes in the in vivo model described herein which is used in conjunction with the antigenic compositions described herein, the following experiments were performed. Briefly, 5 mice per group were treated on day 0, 2, 4, and 6 with either PBS, E. coli colon antigenic compositions, or K. pneumoniae antigenic compositions. On day 7 of the experiment, the mice were sacrificed and a bronchoalveolar lavage was performed. Subsequently the lungs and proximal colon were removed and enzymatically digested. After digestion, the recovered cells were washed and stained with antibodies specific for I-A/I-E FITC (MHC class II; M5/114.15.2); anti-Gr-1 PE (RB6-8C5_; anti-CD11b PerCP-Cy5 (M1/70); anti-CD11c APC (N418). All antibodies were acquired from eBioscience (San Diego, Calif.). The lung cells were counted to determine the total number of cells (the colon was not counted because we did not remove equal amounts of colon between samples). After staining for 20 mins the cells were washed and analyzed by FACS. Each data point shown in corresponding FIG. 5 represents the frequency of CD11b+Gr-1+ cells in the live gate for one mouse. As shown in FIG. 5, treatment with E. coli antigenic compositions leads to an increased frequency of inflammatory monocytes in the colon of treated mice.

Further, and as shown in FIGS. 6A to 6B, when monocytes in the lungs were examined based on the experimental methods detailed herein, it was found that while both E. coli and K. pneumoniae antigenic compositions increase the frequency of monocytes in the lungs of mice, K. pneumoniae antigenic compositions were more effective when counting for total numbers. Referring to FIGS. 6A to 6B, the left-most panel shows the frequency of CD11b+Gr-1+ (inflammatory monocyte) cells in the lungs; the right-most panel shows the total number of CD11b+Gr-1+ cells in the lung.

Example 3: Microbial Prophylaxis in Lungs Example 3a: Klebsiella pneumoniae-Derived Antigenic Formulation (SSI) Protects Against S. pneumoniae Challenge in the Lungs

This Example illustrates that a SSI preparation of whole killed Klebsiella pneumonia administered subcutaneously in mice induced increases in circulating monocytes and provides protection against subsequent bacterial challenge with Streptococcus pneumoniae introduced into the nasopharynx. Streptococcus pneumoniae, or pneumococcus, is a Gram-positive, alpha-hemolytic, aerobic member of the order Lactobacillales. In contrast, Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose-fermenting, facultative anaerobe member of the order Enterobacteriales. These organisms are classified in distinct taxanomic phyla.

C57BL/6 mice were pretreated by subcutaneously injection with placebo or the Klebsiella pneumonia formulation every other day for three weeks, then challenged with 1×10⁹ CFU of S. pneumonia P1547 and monitored for 5 days for signs of clinical infection. On day 5, mice were sacrificed (moribund mice were sacrificed before day 5) and evaluated for bacterial load and immune parameters.

As illustrated in FIG. 7A, mice treated with the Klebsiella pneumonia formulation were protected from S. pneumonia infection (5/5) whereas placebo-treated mice were only partially protected (3/5). Furthermore, TNF and MCP-1 gene expression in lungs of resistant mice (both SSI- and placebo-treated) were higher than placebo-treated mice that succumbed to infection. FIG. 7B illustrates reduced bacterial counts in nasal cavity, lungs, and spleen with the SSI treatment.

Example 3b: Klebsiella pneumoniae-Derived Antigenic Formulation (SSI) Protects Against P. aeruginosa or S. pneumoniae Challenge in the Lungs

Pseudomonas aeruginosa (PA14, 7.8×10⁸ CFU/mouse) was instilled into lungs of 8-10 week old C57131/6 mice pretreated with 30 μL PBS or an SSI formulated from whole killed Klebsiella pneumoniae every other day for 3 weeks, with survival as shown in FIG. 9A. Of the 6 surviving mice at day 5, 3/6 of the PBS treated group had bacteria in the lungs and only 2/10 SSI treated mice had bacteria in the lungs, as shown in FIG. 9B.

Streptococcus pneumoniae (P1542, 4.1×10⁶ CFU/mouse) was instilled into lungs of 8-10 wo C57Bl/6 mice pretreated with 30 μL PBS or an SSI formulated from whole killed Klebsiella pneumoniae every other day for 3 weeks, with survival as shown in FIG. 10.

This Example illustrates that a SSI preparation of whole killed Klebsiella pneumonia administered subcutaneously in mice induces prophylactic antimicrobial activity against infection in the lungs with Pseudomonas aeruginosa or S. pneumoniae. FIGS. 9A to 9B illustrate significantly enhanced survival in a Pseudomonas aeruginosa challenge, and FIG. 10 illustrates enhanced survival in a S. pneumoniae challenge. These data illustrate prophylaxis mediated by an antigenic composition of one lung pathogen, effective against two heterologous lung pathogens.

Example 3c: Comparison of Klebsiella pneumoniae-Derived Antigenic Formulation (SSI) and E. coli-Derived Antigenic Formulation (SSI) in Protecting Against S. pneumoniae or P. aeruginosa Challenge in the Lungs

In this Example, Klebsiella pneumoniae SSI (QBKPN) demonstrated statistically superior efficacy in prophylaxis compared to the E. coli SSI (QBECO), in protecting against S. pneumoniae or P. aeruginosa challenge in the lungs.

For the model treatment of P. aeruginosa, mice were treated with the indicated SSI (0.03 ml/injection) for 14 days, then challenged with Pseudomonas aeruginosa (PA14) by intranasal instillation of 6.0×10⁸ CFU of bacteria. Three days later, lungs were aseptically resected, homogenized, and assessed for bacterial load using Pseudomonas selection agar plates. FIG. 15 shows the results (data are CFU/g of lung (mean+/− std dev), illustrating Klebsiella pneumoniae SSI (QBKPN) demonstrated statistically superior efficacy in prophylaxis compared to the E. coli SSI (QBECO), in protecting against P. aeruginosa challenge in the lungs.

For the model treatment of S. pneumoniae, mice were treated with the indicated SSI (0.03 ml/injection) for 14 days, then challenged with Streptococcus pneumoniae (PA14) by intranasal instillation of 5.0×10⁵ CFU of bacteria. Three days later, lungs were aseptically resected, homogenized, and assessed for bacterial load using Pseudomonas selection agar plates. FIG. 16 shows the results (data are CFU/g of lung (mean+/− std dev), illustrating Klebsiella pneumoniae SSI (QBKPN) demonstrated statistically superior efficacy in prophylaxis compared to the E. coli SSI (QBECO), in protecting against S. pneumoniae challenge in the lungs.

Example 4: Antimicrobial Therapy in GIT

This example illustrates heterologous anti-microbial therapy in a mouse model of inflammatory bowel disease that uses a specific species (NRG 857) of Adherent Invasive E. coli (AIEC) to induce a chronic IBD-like infection in the colon in the 129e strain of mice that have a reduced ability to clear AIEC.

The 129e mice were infected with NRG 857 and treated with either (1) placebo, (2) a whole killed cell formulation of E. coli (SSI-1 in FIG. 8A to 8B), or (3) a whole killed cell formulation of S. enterica, which is an exogenous pathogen which can cause gastrointestinal infection in mice (referred to as SSI-2 in FIG. 8). The results illustrate that the whole killed formulation of E. coli was effective in reducing counts of NRG 857 in colon tissue (300 times less than placebo treated mice—note that the graph is a logarithmic scale) and feces of 129e mice. In addition, the S. enterica formulations also exhibited some anti-microbial activity against the heterologous AIEC infection.

In accordance with one aspect of the invention, macrophage defect or deficiency, leading to a reduced ability to clear bacterial infection and necrotic debris, may be the underlying trigger for some pathologies associated with microbial infections. This has for example been postulated to be the case in Crohn's disease. Aspects of the invention accordingly involve the induction of organ specific macrophage recruitment and activation, resulting in clearance of bacterial infection.

Example 5: Antimicrobial Prophylaxis in Peritoneal Cavity

In a first aspect, this example illustrates that a whole killed E. coli SSI formulation is effective in protecting against a S. enterica. As illustrated in FIGS. 11A to 11B, SSI treated mice had significantly fewer bacteria in the spleen than saline treated mice (p<0.0001), and SSI treated mice had less peak weight loss than saline treated mice (p<0.02).

In an alternative aspect, this Example illustrates that two alternative whole killed E. coli SSI formulations (QBECO and QBECP), made from different strains of E. coli, are effective in a mouse model in protecting against a S. enterica challenge, with levels of prophylaxis that compare favourably to prior administration of an antigenic S. enterica formulation (QBSEN). In this Example, mice were pre-treated by skin injection with the alternative formulations, QBECO, QBECP (SSI from urologic E. coli), or QBSEN (each given every other day for 3 weeks) and then infected with S. enterica. Survival was improved for all treatments, with QBECP providing comparable benefit to QBSEN, as illustrated in FIG. 12A. Mice were sacrificed, and colony counts in the spleen were quantitated and found to be lower in all the treated groups, as illustrated in FIG. 12B. Similarly, weight loss was found to be lower in all the treated groups, as illustrated in FIG. 12C.

In a further illustration of targeted and optimized intraperitoneal (IP) prophylaxis, mice were treated with alternative SSIs, QBKPN and QBECO, or vehicle; then challenged with Salmonella enterica (typhimurium) by IP instillation of 1.0×10⁶ CFU of bacteria. Three days later, spleens were aseptically resected, homogenized, and assessed for bacterial load on Hektoen enteric agar plates. FIG. 19 illustrates that both QBECO and QBKPN are protective, as measured by bacterial load in the spleen, and counts in mice treated with QBECO are significantly lower than mice treated with QBKPN (data are CFU/ml (mean+/− std dev).

Example 6: Antimicrobial Prophylaxis in Skin

This example illustrates targeted heterologous anti-microbial therapy in a mouse model of skin infection, illustrating the improved efficacy of antigenic formulations derived from microbial pathogens of the target tissue. Mice were treated with selected antigenic formulations of the invention (0.03 ml/injection) for 14 days, then challenged with Pseudomonas aeruginosa (PA14) by intradermal injection of 6.5×10⁵ CFU. Three days later, skin was aseptically resected, homogenized, and assessed for bacterial load using Pseudomonas selection agar plates. FIG. 13 illustrates that QBSAU (S. aureus-derived SSI) protects against P. aeruginosa challenge in skin, with significantly reduced bacterial counts following QBSAU, compared to QBKPN (which is not a skin pathogen in the murine model). A repeat iteration of the foregoing procedure replicated the results, as shown in FIG. 14 (n=8 mice/group).

Example 7: Antimicrobial Prophylaxis in Geriatric Model Aged Mice

This example illustrates targeted heterologous anti-microbial therapy in a geriatric mouse model of lung infection, showing that Klebsiella pneumoniae SSI (QBKPN) protects against S. pneumoniae challenge in lungs of aged mice. FIGS. 17A to 17B show that the survival benefit for old mice, FIG. 17B, was more pronounced than for young mice, FIG. 17A. FIG. 18 illustrates corroborative data, showing that QBKPN reduces weight loss in aged mice, following challenge with S. pneumoniae, and this benefit is greater for aged mice compared to young mice.

Example 8: Antiviral Prophylaxis in Lungs

In this Example, mice wherein injected with QBKPN or QBECO SSI, or vehicle for 14 days, in accordance with the protocol outlined in other Examples. Mice were then challenged intranasally with murine herpes virus 68 (MHV68), a virus that is genetically modified to drive a luciferase reporter. MHV68 is a model viral pathogen for studying lung infection. Three days after challenge, mice were injected with 300 micrograms of luciferin; 10 min later, mice were sacrificed, lungs resected, and imaged with a Xenogen IVIS imager. Luminescence in the lungs is an indicator of active viral infection and replication.

Pretreatment of mice with QBKPN SSI dramatically diminished the luminescence signal in lungs of mice, providing evidence of antiviral prophylaxis in lungs using targeted antigenic bacterial formulations. The same dramatic result was not observed for QBECO.

OTHER EMBODIMENTS

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. In some embodiments, the invention excludes steps that involve medical or surgical treatment. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. 

What is claimed is:
 1. A method of modulating an immune system in a vertebrate host for the prophylactic [or therapeutic]treatment of infection by a first microbial pathogen in a target tissue, the method comprising: administering to the vertebrate host an effective amount of an antigenic formulation comprising antigenic determinants specific for a second heterologous microbial pathogen, wherein: the antigenic determinants are selected from the group consisting of whole cells or cell wall extracts; the first microbial pathogen is a microbial lung pathogen that is not a Klebsiella pneumonia, Streptococcus pneumonia, Pseudomonas aeruginosa or a viral lung pathogen, and the second heterologous microbial pathogen is a Klebsiella pneumonia, and the target tissue is the lung; and, wherein the administering comprises administering successive doses of the formulation at a dosage interval of at least one hour, so that two or more doses are administered over a period from 2 days to 1 month, over a dosage duration of at least one week.
 2. The method of claim 1, wherein the first microbial pathogen is Moraxella catarrhalis, Mycoplasma pneumoniae, or Haemophilus influenza.
 3. The method of claim 1, wherein the host is a human patient.
 4. The method of claim 2, wherein the host is a human patient.
 5. The method of claim 1, wherein the antigenic determinants comprise whole cells.
 6. The method of claim 2, wherein the antigenic determinants comprise whole cells.
 7. The method of claim 3, wherein the antigenic determinants comprise whole cells.
 8. The method of claim 4, wherein the antigenic determinants comprise whole cells.
 9. The method of claim 5, wherein the whole cells are killed.
 10. The method of claim 6, wherein the whole cells are killed.
 11. The method of claim 7, wherein the whole cells are killed.
 12. The method of claim 8, wherein the whole cells are killed.
 13. The method of claim 5, wherein the whole cells are attenuated.
 14. The method of claim 6, wherein the whole cells are attenuated.
 15. The method of claim 7, wherein the whole cells are attenuated.
 16. The method of claim 8, wherein the whole cells are attenuated.
 17. The method of claim 1, wherein the antigenic determinants comprise cell wall extracts.
 18. The method of claim 2, wherein the antigenic determinants comprise cell wall extracts.
 19. The method of claim 3, wherein the antigenic determinants comprise cell wall extracts.
 20. The method of claim 4, wherein the antigenic determinants comprise cell wall extracts. 